Methods for screening antibodies

ABSTRACT

The invention provides methods for making antibody conjugates for use in antibody screening assays and antibody conjugates produced by the claimed methods.

This application is a continuation of U.S. patent application Ser. No.13/581,236 filed Aug. 24, 2012, which is the national stage filing under35 U.S.C. §371 of International Application No. PCT/US2011/026534 filedFeb. 28, 2011, which claims the benefit of U.S. Provisional App. No.61/309,725 filed Mar. 2, 2010 and U.S. Provisional App. No. 61/323,433filed Apr. 13, 2010, each of which is incorporated by reference in itsentirety for all purposes.

BACKGROUND OF THE INVENTION

The activity of antibody-drug conjugates (ADCs) on cancer cells can beaffected by a multitude of factors, such as binding affinity, rate ofinternalization, subcellular trafficking, and efficient drug releasewithin the target cell population. Consequently, the properties of anideal antibody for drug delivery are not necessarily the same as thosefor a therapeutic unconjugated antibody. Furthermore, indirect assaysinvolving the use of secondary antibodies to screen for optimal ADCs canbe misleading, since crosslinking on the cell surface can lead toaltered downstream events, and the affinity of the secondary antibodyconstrains the dynamic range of the assay. When seeking candidateantibodies directed against a novel antigen for ADC therapy, it istherefore most desirable to screen a large antibody panel in the form ofADCs and evaluate their cytotoxic activities, since these resultsprovide a direct measurement of parameters that can affect cytotoxicactivity. However, when dealing with microgram quantities of a largenumber of antibodies as is typical of an antibody discovery campaign,the yields from conventional conjugation methodologies are limiting. Aneed exists for improved methods of screening antibodies for use asADCs. This present invention addresses this and other needs.

SUMMARY OF THE INVENTION

The invention provides methods for making antibody conjugates for use inantibody screening assays and antibody conjugates produced by theclaimed methods.

In some embodiments, the methods comprise the steps of providing a firstand second antibody-containing sample wherein the first and secondantibody-containing sample vary with respect to antibody quantity andantibody sequence provided that substantially all of the antibodypresent in the first sample is of the same sequence and substantiallyall of the antibody present in the second sample is of the samesequence; immobilizing the antibodies on a solid support to provide afirst and second sample comprising immobilized antibodies; fullyreducing the reducible disulfide bonds of the immobilized antibodies toprovide a first sample comprising reduced immobilized antibodies andsecond sample comprising reduced immobilized antibodies, wherein thereduction is selective for the reducible disulfide bonds; reacting thereduced immobilized antibodies with capping agent, drug or drug-linker,and optionally a detection agent to provide immobilized antibodyconjugates, wherein the capping agent, drug or drug-linker, and optionaldetection agent selectively react with reactive thiols, the cappingagent, drug or drug-linker, and optional detection agent are provided inmolar excess, and the ratio of capping agent, drug or drug linker, andoptional detection agent is selected so as to achieve a desired level ofdrug loading; and eluting the antibody conjugates to provide a firstsample of free antibody conjugates and second sample of free antibodyconjugates.

In some embodiments, the methods comprise the steps of providing aplurality of samples of unpurified hybridoma supernatant comprisingunquantified antibody produced from a plurality of hybridoma clones,wherein the plurality of samples vary with respect to antibody quantityand antibody sequence provided that, in a majority of the plurality ofthe samples, substantially all of the antibody present in each sample isfrom a single hybridoma clone; immobilizing the unquantified antibodieson a solid support to provide a plurality of samples comprisingimmobilized antibodies; fully reducing the interchain disulfides of theimmobilized antibodies to provide a plurality of samples comprisingreduced immobilized antibodies; reacting the reduced immobilizedantibodies with capping agent, drug or drug-linker, and a detectionagent to provide immobilized antibody conjugates, wherein the cappingagent, drug or drug-linker, and detection agent selectively react withreactive thiols, the capping agent, drug or drug-linker, and optionaldetection agent are provided in molar excess, and the ratio of cappingagent, drug or drug linker and detection agent is selected so as toachieve a desired level of drug loading; and eluting the antibodyconjugates from the solid supports to provide a plurality of antibodyconjugate compositions.

In some embodiments, the methods comprise the steps of providing aplurality of antibody containing samples that vary with respect toantibody quantity and antibody sequence provided that, in a majority ofthe plurality of the antibody-containing samples, substantially all ofthe antibody present in a single sample is of the same sequence;immobilizing the antibodies on a solid support to provide a plurality ofsamples comprising immobilized antibodies; fully reducing the reducibledisulfide bonds of the immobilized antibodies to provide a plurality ofsamples comprising reduced immobilized antibodies, wherein the reductionis selective for reducible disulfide bonds; reacting the reducedimmobilized antibodies with capping agent, drug or drug-linker, andoptionally a detection agent to provide a plurality of samplescomprising immobilized antibody conjugates, wherein the capping agent,drug or drug-linker, and optional detection agent selectively react withreactive thiols, the capping agent, drug or drug-linker, and optionaldetection agent are provided in molar excess, and the ratio of cappingagent, drug or drug linker; and optional detection agent is selected soas to achieve a desired level of drug loading; and eluting the antibodyconjugates to provide a plurality of antibody conjugate compositionscomprising free antibody conjugates.

In some embodiments, the methods comprise the steps of providing aplurality of antibody containing samples that vary with respect toantibody quantity and antibody sequence provided that, in a majority ofthe plurality of the antibody containing samples, substantially all ofthe antibody present in a single sample is of the same sequence;immobilizing the antibodies on a solid support to provide a plurality ofsamples comprising immobilized antibodies; fully reducing the reducibledisulfide bonds of the immobilized antibodies to provide a plurality ofsamples comprising reduced immobilized antibodies, wherein the reductionis selective for reducible disulfide bonds; reacting the reducedimmobilized antibodies with capping agent, and a detection agent toprovide a plurality of samples comprising immobilized antibodyconjugates, wherein the capping and detection agent selectively reactwith reactive thiols, the capping agent, and detection agent areprovided in molar excess, and the ratio of capping agent and detectionagent is selected so as to achieve a desired level of detection agentand/or capping agent loading; and eluting the antibody conjugates toprovide a plurality of antibody conjugate compositions comprising freeantibody conjugates.

In some embodiments, the methods comprise the steps of providing aplurality of antibody containing samples that vary with respect toantibody quantity and antibody sequence provided that, in a majority ofthe plurality of the antibody-containing samples, substantially all ofthe antibody present in a single sample is of the same sequence;immobilizing the antibodies on a solid support to provide a plurality ofsamples comprising immobilized antibodies; fully reducing the reducibledisulfide bonds of the immobilized antibodies to provide a plurality ofsamples comprising reduced immobilized antibodies, wherein the reductionis selective for reducible disulfide bonds; reacting the reducedimmobilized antibodies with capping agent, drug or drug-linker, andoptionally a detection agent to provide a plurality of samplescomprising immobilized antibody conjugates, wherein the capping agent,drug or drug-linker, and optional detection agent selectively react withreactive thiols, the capping agent, drug or drug-linker, and optionaldetection agent are provided in molar excess, and the ratio of cappingagent, drug or drug linker; and optional detection agent is selected soas to achieve a desired level of drug loading; eluting the antibodyconjugates to provide a plurality of antibody conjugate compositionscomprising free antibody conjugates; assaying for an activity of theantibody conjugates; and selecting an antibody of the basis of theoutcome of the assay.

In some embodiments, the methods comprise the steps of providing aplurality of antibody containing samples that vary with respect toantibody quantity and antibody sequence provided that, in a majority ofthe plurality of the antibody containing samples, substantially all ofthe antibody present in a single sample is of the same sequence;immobilizing the antibodies on a solid support to provide a plurality ofsamples comprising immobilized antibodies; fully reducing the reducibledisulfide bonds of the immobilized antibodies to provide a plurality ofsamples comprising reduced immobilized antibodies, wherein the reductionis selective for reducible disulfide bonds; reacting the reducedimmobilized antibodies with capping agent, and a detection agent toprovide a plurality of samples comprising immobilized antibodyconjugates, wherein the capping and detection agent selectively reactwith reactive thiols, the capping agent, and detection agent areprovided in molar excess, and the ratio of capping agent and detectionagent is selected so as to achieve a desired level of detection agentand/or capping agent loading; eluting the antibody conjugates to providea plurality of antibody conjugate compositions comprising free antibodyconjugates; assaying for an activity of the antibody conjugates; andselecting an antibody of the basis of the outcome of the assay.

These and other aspects of the present invention may be more fullyunderstood by reference to the following detailed description,non-limiting examples of specific embodiments, and the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. This figure provides an overlay of hydrophobic interactionchromatograms of a murine IgG1 in its unconjugated form (dashed), fullyreduced and conjugated with mcMMAF in solution (heavy solid line), andfully reduced and conjugated with mcMMAF while immobilized on Protein Gsepharose (light solid line).

FIG. 2. This figure illustrates the mole fraction of mcMMAF in anexemplary reaction mixture comprising mcMMAF and N-ethyl maleimidenecessary in order to achieve a select drug loading on a murine IgG1murine immobilized on protein G and fully reduced with excesstris(2-carboxyethyl)phosphine.

FIG. 3. The figure provides a sample PLRP chromatogram of an antibodydrug conjugate illustrating the distribution of mcMMAF and NEM on theheavy and light chains of the antibody. The hydrophobicity of the drugresults in later retention times for species with more drug; the numberof drugs for each species is indicated.

FIG. 4. This figure demonstrates the fluorescence output of drug-AlexaFluor® 647 conjugates as a function of fluorophore loading. The numberof fluorophores per antibody is plotted on the x axis and fluorescenceis plotted on the y axis. Fluorescence increases rapidly to a maximumvalue when loading is about 2.5 to 3 fluorophores per antibody, thendecreases with further loading.

FIG. 5. This figure provides the ratio of the absorbance at 650 nm to280 nm plotted as a function of Alexa Fluor® 647 loading level in mixedfluorophore-mcMMAF antibody conjugates.

FIG. 6. This figure demonstrates the consistency of Alexa Fluor® 647loading across 65 samples. The fluorophore loading was determined byobtaining the 650 nm/280 nm absorbance ratio of each antibody conjugatesample and referring back to FIG. 5 to determine the fluorophore loadingassociated with the absorbance ratio.

FIG. 7. This figure provides a PLRP chromatogram of an mcMMAF-AF647-NEMmixed conjugate. The antibody has 5 reducible disulfides. This figureprovides an overlay of two analytical wavelengths. The 280 nm wavelengthrepresented by a light solid line detects all of the peaks containingprotein and the 620 nm wavelength represented by a heavy solid linedetects all of the peaks containing at least one Alexa Fluor® 647.

FIG. 8. This figure illustrates the consistency of mcMMAF loading across34 samples.

FIG. 9. This figure provides a PLRP chromatogram of an mcMMAF-AF647-NEMmixed conjugate. The antibody has 6 reducible disulfides (e.g., a murineIgG2b). The 280 nm wavelength is represented by a light solid line andthe 620 nm wavelength is represented by a heavy solid line.

FIG. 10. This figure provides an exemplary scheme for plate-based solidphase synthesis of ADCs.

FIG. 11. This figure provides an exemplary scheme for application ofsolid phase conjugation technology to the discovery of ADCs withdesirable properties.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” as used herein refers to (a) immunoglobulinpolypeptides and immunologically active portions of immunoglobulinpolypeptides, i.e., polypeptides of the immunoglobulin family, orfragments thereof or (b) conservatively substituted derivatives of suchimmunoglobulin polypeptides or fragments that immunospecifically bind toa target antigen. The antibodies (including antibody fragments) for usein the present invention contain (i) an antigen binding site thatimmunospecifically binds to a target antigen, (ii) at least onereducible disulfide bond (e.g., interchain disulfide bond) and (iii) adomain capable of reversibly binding to a solid phase. In someembodiments, an antibody will comprise a full length Fc region andbinding to the solid phase will be through the Fc region. In someembodiments, an antibody will comprise one or more Fc domains of anantibody and binding to the solid phase will be through the one or moreFc domains. In some embodiments, the domain capable of reversiblybinding to a solid phase will not be a Fc region, but will be a domainengineered on the antibody, such as, for example, an affinity tag. Theterm antibody includes antibodies that are non-fucosylated or havereduced core fucosylation. Antibodies are generally described in, forexample, Harlow & Lane, Antibodies: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1988). The basic unit of an intact antibodystructure is a complex of four polypeptides--two identical low molecularweight (“light”) chains and two identical high molecular weight(“heavy”) chains, linked together by both non-covalent associations andby disulfide bonds. The class and subclass of an antibody is itsisotype. Antibodies can be, for example, in their natural tetramericform (2 light chains and 2 heavy chains) and can be of any of the knownisotypes IgG, IgA, IgM, IgD and IgE and their subtypes, for example,human IgG1, IgG2, IgG3, IgG4 and mouse IgG1, IgG2a, IgG2b, and IgG3. Theantibodies are preferably monoclonal.

In the context of an antibody, the term “reducible disulfide bond”refers to a disulfide bond that is (i) reducible while the antibody isreversibly bound to a solid support, and (ii) reducible under mildreducing conditions. Mild reducing conditions are those conditions thatgenerally do not cause any substantial denaturation of the antibody andgenerally do not affect the antigen binding affinity of the antibody. Anexample of mild reducing conditions is reduction under aqueousconditions at near neutral pH with a weak reducing agent. An example ofweak reducing agents are TCEP (tris(2-carboxyethyl)phosphine) and DTT(dithiothreitol). Accordingly, one example of mild reducing conditionsis reduction in an excess of TCEP or DTT at a temperature of about 5° C.to about 37° C. and a pH of from about 5 to 8. Because organiccosolvents can substantially denature proteins, if organic cosolventsare to be used in the denaturation and/or subsequent conjugation steps,it should be a minimal amount of cosolvents (e.g., less than 20%,preferably less than 15%, 10%, or even 5%) such that substantialdenaturation of the antibody does not occur. Typically, the reducibledisulfide bonds are those that are solvent accessible, i.e., not buriedwithin the folded domains of the antibody. (The skilled artisan willunderstand that when reducing the reducible disulfide bonds of apopulation of antibodies within a sample according to the methodsdescribed herein, there may be a minor amount of antibodies that dobecome irreversibly denatured (e.g., generally less than 10%, even lessthan 5% or 3%)). Typically, in an antibody, a disulfide bond is presentas a result of the oxidation of the thiol (—SH) side groups of twocysteine residues. These residues may lie on different polypeptidechains (interchain), or on the same polypeptide chain (intrachain). As aresult of the oxidation, a disulfide bond (—S—S—) is formed between thebeta carbons of the original cysteine residues. Treatment of thedisulfide bond with a reducing agent causes reductive cleavage of thedisulfide bonds to generate two free thiol groups, i.e., reactivethiols. In some embodiments, the reducible disulfide bond is naturallyoccurring. In some aspects, the term “reducible disulfide bond” refersto the naturally occurring interchain disulfide bonds of an antibody. Insome embodiments, a sulfhydryl group(s) is chemically introduced intothe antibody. Suitable methods for introducing sulfhydryl groups includerecombinant DNA technology. Sulfhydryl groups can be introduced into anantibody, for example, within the antibody or at the carboxy-terminus.Because it is preferable that the methods described herein do notinterfere with the antigen binding activity of the resultant antibodyconjugates, it is preferable that introduced sulfhydryl groups beintroduced at a site other than the antigen binding site of theantibody. Preferably introduced sulfhydryl groups are introduced at asite other than the heavy or light chain variable regions, e.g.,preferably in the constant region of an antibody. In some embodiments, acysteine residue is engineered into an antibody. The sulfhydryl group ofthe cysteine will typically form a disulfide bond that can then bereduced using the methods described herein.

In the context of a fusion protein, the term “reducible disulfide bond”refers to a disufide bond of a fusion protein that is (i) reduciblewhile the fusion protein is reversibly bound to a solid support, and(ii) reducible under mild reducing conditions. For a fusion protein tobe of use in the present methods, it should remain generally intactfollowing reduction of the reducible disulfide bond(s). An example ofmild reducing conditions is reduction under aqueous conditions at nearneutral pH with a weak reducing agent. In some preferred embodiments,the reducible disulfide bond will be in the Ig domain of the fusionprotein. In some embodiments, the disulfide bond is naturally occurringand refers to the naturally occurring interchain disulfide bonds of theIg domain of the fusion protein. In some embodiments, a sulfhydrylgroup(s) is chemically introduced into Ig domain of the fusion protein.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single cell clone, including any eukaryotic or prokaryotic cellclone, or a phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology.

The term “Fc region” refers to a constant region of an antibody, e.g., aC_(H)1-hinge-C_(H)2-C_(H)3 domain, optionally having a C_(H)4 domain, ora conservatively substituted derivative of such an Fc region.

The term “Fc domain” refers to the constant region domain of anantibody, e.g., a C_(H)1, hinge, C_(H)2, C_(H)3 or C_(H)4 domain, or aconservatively substituted derivative of such an Fc domain.

An “antigen” is a molecule to which an antibody specifically binds.

A “cytotoxic agent” refers to an agent that has a cytotoxic and/orcytostatic effect on a cell. A “cytotoxic effect” refers to thedepletion, elimination and/or the killing of a target cell(s). A“cytostatic effect” refers to the inhibition of cell proliferation.

The term “interchain disulfide bond,” in the context of an antibody,refers to a disulfide bond between two heavy chains, or a heavy and alight chain of an antibody.

As used herein, “free antibody conjugates” refers to antibody conjugatesthat are not immobilized on a solid support, e.g., antibodies that havebeen released from a solid support.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediaminehaving the general formula shown immediately following:

The abbreviation “MMAE” refers to monomethyl auristatin E having thegeneral formula shown immediately following:

The abbreviation “MMAF” refers todovaline-valine-dolaisoleunine-dolaproine-phenylalanine having thegeneral formula shown immediately following:

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid. The abbreviation “AEVB”refers to an ester produced by reacting auristatin E with benzoylvalericacid.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a molecule ormacromolecule. Acid addition salts can be formed with amino groups.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′ methylene bis-(2-hydroxy 3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

General

A method of directly screening antibodies on the basis of theirperformance as ADCs or as unconjugated antibodies (i.e., nakedantibodies) has been invented. A labeling technique has been developedthat is insensitive to the concentration of antibody present in a sampleand applicable to small amounts of antibody allowing for a comparison ofthe activities of individual antibodies of a heterogenous population ofantibodies.

The screening assay is useful for identifying antibodies with desiredcharacteristics. The antibodies can be generated though any techniqueknown in the art for generating antibodies provided that the antibodiesto be screened comprise (i) an antigen binding site thatimmunospecifically binds to a specific antigen, (ii) at least onereducible disulfide bond (e.g., interchain disulfide bond) and (iii) adomain capable of binding to a solid phase.

In one aspect of the invention, a plurality of antibody-containingsamples are provided. The phrase “a plurality of samples” refers to twoor more samples. Because the methods provided herein are ideally suitedfor high throughput screening, in one aspect of the invention, themethods are performed simultaneously on at least tens or at leasthundreds of samples. One of the strengths of the methods provided hereinis that a comparison between antibodies can be made even though theantibody-containing samples may not contain the same quantity ofantibody. Accordingly, in one aspect, the samples vary with respect toantibody quantity and with respect to antibody sequence. For example, inone aspect, a first sample will comprise a first antibody at a firstquantity and a second sample will comprise a second antibody at a secondquantity. The first and second quantities will vary and the first andsecond antibodies will vary. In embodiments wherein it is desirable tocompare antibodies that target the same antigen, the antibodies willimmunospecifically bind to the same antigen. For purpose ofclarification, the phrase “wherein the plurality of samples vary withrespect to antibody quantity and antibody sequence” does not requirethat all of the samples within a plurality of samples vary with respectto antibody quantity and antibody sequence, only that there is certainlevel of heterogeneity between samples. Although there is a variance inantibody sequence (e.g., a first sample will contain a differentantibody than a second sample), it is preferable that a single samplecontain one antibody, i.e., that the antibody present in a single sampleis of the same sequence. The phrase “substantially all of the antibodypresent in a single sample is of the same sequence” reflects thepreference that a single sample contain one antibody with therecognition that, in some samples, there may be some contamination withanother antibody. Preferably, in those samples that have somecontamination with another antibody, there is less than 30%, preferablyless than 20%, preferably less than 15%, more preferably less than 10%,and even more preferably less than 5%, less than 4%, or less than 3% ofcontamination with another antibody. In preferred embodiments, themajority of antibody-containing samples (greater that 50% of samples andeven more preferably greater than 60%, greater than 70%, greater than75%, or even greater than 80% of the samples) in a plurality ofantibody-containing samples contain one antibody with no or minoramounts of contamination with another antibody (e.g., less than 15%,preferably even less than 10% or less than 5% contamination with anotherantibody). In some preferred embodiments, a majority of theantibody-containing samples will comprise antibodies thatimmunospecifically bind to the same antigen.

The antibodies to be screened using the present methods can be targetedto any antigen. In exemplary embodiments, an antibody to be screened bythe present methods will immunospecifically bind to an antigen selectedfrom CD19, CD20, CD21, CD22, CD30, CD33, CD38, CD40, CD70, CD74, CD83,CD133, CD138, CD200, or CD276. In other embodiments, the antibody willimmunospecifically bind to BMPR1B, LAT1 (SLC7A5), STEAP1, MUC16, MUC1,megakaryocyte potentiating factor (MPF), Napi3b, Sema 5b, PSCA hlg, ETBR(Endothelin type B receptor), STEAP2, TrpM4, CRIPTO, CD21, CD79a, CD79b,FcRH2, HER2, HER3, HER4, NCA, MDP, IL20Rα, Brevican, Ephb2R, ASLG659,PSCA, PSMA, TMPRSS2, TMPRSS4, GEDA, BAFF-R, CXCRS, HLA-DOB, P2X5, CD72,LY64, FCRH1, VEGF, PLAC1, VEGFR1, VEGFR2, or IRTA2. In otherembodiments, the antibody will immunospecifically bind to CD2, CD3,CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD23, CD25, CD28, CD29,CD30, CD32, CD4OL, CD51, CD52, CD54, CD56, CD70, CD80, CD123, CD133,CD138, CD147, CD227, or CD276. In other embodiments, the antibody willimmunospecifically bind to IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6,IL-6R, IL-8, IL-12, IL-15, IL-18, or IL-23. In other embodiments, theantibody will immunospecifically bind to a protein from the solutecarrier family of proteins (e.g., solute carrier family 44, member 4(protein encoded by SLC44A4 gene) or solute carrier family 34, member 2(protein encoded by the SLC34A2 gene)); LIV-1 (protein encoded bySLC39A6 gene); protein from the SLAM family of proteins (e.g., SLAMfamily members 1, 2, 3, 4, 5, 6, 7, 8 or 9); protein from the mucinfamily of proteins (e.g., MUC1, MUC2, MUC3, MUC4, MUCS, MUC6, MUC7,MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, or MUC16); proteinfrom the STEAP family of proteins (e.g., STEAP1, STEAP2, STEAP3 orSTEAP4); a protein from the tumor necrosis factor receptor family (e.g.,TNF-RI, TNF-RII, DR1, DR2, DR3, DR4, DRS); MN protein; mesothelinprotein; protein encoded by the Slitrk family of proteins (e.g.,SLITRK1, SLITRK2, SLITRK3, SLITRK4, SLITRKS, or SLITRK6), or a proteinencoded by the GPNMB gene.

The antibody-containing samples can be generated in many different ways.There are many techniques known in the art for generating antibodies.For example, antibodies that are useful in the present methods can beproduced by recombinant expression techniques, phage display technique,from hybridomas, from myelomas, from other antibody expressing mammaliancells, and from combinations thereof. Antibodies to be used in thepresent invention can be of any species (e.g., human, murine, rat) andcan be of mixed species, e.g., chimeric. Antibodies to be used in thepresent invention can comprise full length variable regions or fragmentsthereof.

A variety of mammalian cells and cell lines can be utilized to expressan antibody. For example, mammalian cells such as Chinese hamster ovarycells (CHO) (e.g., DG44, Dxbll, CHO-K, CHO-K1 and CHO-S) can be used. Insome embodiments, human cell lines are used. Suitable myeloma cell linesinclude SP2/0 and IR983F and human myeloma cell lines such as Namalwa.Other suitable cells include human embryonic kidney cells (e.g.,HEK293), monkey kidney cells (e.g., COS), human epithelial cells (e.g.,HeLa), PERC6, Wil-2, Jurkat, Vero, Molt-4, BHK, and K6H6. Other suitablehost cells include YB2/0 cells.

Any antibody generating techniques can be used to generate theantibody-containing samples described herein provided that theantibodies generated can be immobilized on a solid support and containat least one reducible disulfide bond. In some embodiments, the antibodywill be generated by a method known in the art and will be modified inorder to place it in condition for use in the present methods. Forexample, antibodies generated by phage display or other methods can bemodified to contain an affinity tag and/or can be reformatted to expressa Fc region. For an overview of phage display technology for producingantibodies, see Schmitz et al., 2000, Placenta 21, supplement A,S106-112. See also Lightwood et al., 2006, Journal of ImmunologicalMethods 316, 133-143.

In some aspects, the antibodies to be assayed are generated using wellknown hybridoma techniques. For example, in some embodiments, the hostcells are from a hybridoma. Hybridoma techniques are generally discussedin, for example, Harlow et al., Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 2nd ed., 1988); and Hammerling, et al.,In Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier,N.Y., 1981). Antibodies can also be generated using immortal orconditionally immortal cell lines other than hybridoma cell lines,including, for example, antibodies generated from conditionally immortalcell lines from H-2K^(b)-tsA58 mice (Pasqualinie and Arap, PNAS, 2004,101(1), 257-259). These technologies can be used to generate fullyrodent, chimeric rodent-human, or human antibodies. For example, for anoverview of a technology for producing human antibodies from immunizedtransgenic mice using hybridoma technology, see Lonberg and Huszar,1995, Int. Rev. Immunol. 13:65-93.

In addition, companies such as Amgen, Inc. (Thousand Oaks, Calif.) andBMS (Princeton, N.J.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatreferenced above. Completely human antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of atarget polypeptide. Monoclonal antibodies directed against the antigencan be obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93).

The present methods do not require a purification step prior to antibodyimmobilization on a solid support. In some aspects, the antibodyprovided in the antibody-containing sample is not purified. In someembodiments, unpurified cell culture supernatant or unpurifiedconditioned media is provided as the antibody-containing sample. Forexample, in some embodiments wherein hybridoma technology is used togenerate antibodies, the antibody-containing samples are unpurifiedhybridoma supernatant samples. In some aspects, the supernatant samplesvary with respect to antibody quantity and antibody sequence. It ispreferable that a single hybridoma supernatant sample contain antibodyfrom a single hybridoma clone, although antibody-containing samples cancontain contamination with other antibodies. Methods of picking clonalpopulations from hybridomas are known in the art as are methods ofgenerating hybridoma supernatant. For example, in one aspect, newlyfused hybridomas are plated in semi-solid media (e.g,. methylcellulose)with a selective medium (e.g., a medium that promotes the survival andproliferation of hybridoma cells and the elimination of non-fused Bcells and myeloma cells. Examples of such a medium include onecontaining hypoxanthine, aminopterin and thymidine). ClonalIgG-producing colonies are selected and placed in invididual wellscontaining media to support cell line expansion and antibody production.The resulting hybridoma supernatant can be assayed by the presentmethods. In another aspect, hybridoma cells are cloned using a limiteddilution approach. In some embodiments, prior to immobilization andconjugation, the unpurified hybridoma culture supernatants are screenedfor the presence of antibodies with desired antigen specificity. In someembodiments, from about 1 ml to about 5 mls of hybridoma supernatant isprovided.

In some embodiments, the unpurified cell culture supernatant is otherthan hybridoma supernatant, e.g., CHO cell culture supernatant (e.g.,DG44, Dxb11, CHO-K1 and CHO-S cell lines), or other cell culturesupernatant.

In some embodiments, the antibody in the antibody-containing samples isproduced in culture media lacking endogenous IgG, and, in particular,culture media lacking bovine IgG. In some embodiments, the culturemedium is depleted of endogenous IgG prior to use (see, for example,example 8). Suitable culture media include those containing, forexample, salts, carbon source (e.g., sugars), nitrogen source, aminoacids, trace elements, antibiotics, selection agents, and the like, asrequired for growth. Commerically available media as well ascommercially available cloning media, including IgG depleted cloningmedia can be used. The culture conditions, such as temperature, pH, andthe like, will be apparent to the ordinarily skilled artisan.

The present methods use a solid support for conjugation of theantibodies to a desired chemical entity. Because the present methods areperformed in solid phase and not in solution, the present methods can beperformed with samples that contain very small amounts (e.g., 1 to 500μg) of antibody. In some embodiments, there will be from 1 μg to 100 μg,from 1 μg to 50 μg, from 1 μg to 20 μg, from 3 μg to 100 μg, from 3 μgto 50 μg, from 3 μg to 20, from 5 μg to 100 μg, from 5 μg to 50 μg, from5 μg to 20 μg of antibody present in a single sample. In one aspect, atleast one of the samples in a plurality of samples will have from 1 μgto 100 μg, from 1 μg to 50 μg, from 1 μg to 20 μg, from 3 μg to 100 μg,from 3 μg to 50 μg, from 3 μg to 20, from 5 μg to 100 μg, from 5 μg to50 μg, from 5 μg to 20 μg of antibody present.

A solid support refers to an insoluble, functionalized material to whichthe antibodies can be reversibly attached, either directly orindirectly, allowing them to be separated from unwanted materials, forexample, excess reagents, contaminants, and solvents. Examples of solidsupports include, for example, functionalized polymeric materials, e.g.,agarose, or its bead form Sepharose®, dextran, polystyrene andpolypropylene, or mixtures thereof; compact discs comprisingmicrofluidic channel structures; protein array chips; pipet tips;membranes, e.g., nitrocellulose or PVDF membranes; and microparticles,e.g., paramagnetic or non-paramagnetic beads. In some embodiments, anaffinity medium will be bound to the solid support and the antibody willbe indirectly attached to solid support via the affinity medium. In oneaspect, the solid support comprises a protein A affinity medium orprotein G affinity medium. A “protein A affinity medium” and a “proteinG affinity medium” each refer to a solid phase onto which is bound anatural or synthetic protein comprising an Fc-binding domain of proteinA or protein G, respectively, or a mutated variant or fragment of anFc-binding domain of protein A or protein G, respectively, which variantor fragment retains the affinity for an Fc-portion of an antibody.

The present methods comprise a step of immobilizing antibody on a solidsupport to provide immobilized antibodies. In some embodiments, thesolid support will have the capacity to bind more antibody than theamount present in the antibody-containing sample or, in other words, theamount of antibody bound to the solid support following theimmobilization step will be less than the capacity of the solid support.Because the samples generally vary with respect to antibody quantity,there will be corresponding variability in the amount of immobilizedantibody from one sample as compared to another.

In some other embodiments, it might be desirable to limit the quantityof bound antibody and the solid support will only have the capacity tobind up to a certain amount of antibody (e.g., up to 5 μg, up to 10 μg,or up to 15 μg of protein). In these embodiments, although there will bea limit as to the maxium amount of antibody that can be bound to thesolid support, there may still be variability in the amount ofimmobilized antibody in one sample as compared to another. This isbecause one or more of the samples might contain a small quantity ofantibody, less than the maximum loading capacity of the solid support.One approach for preparing a solid support that has limited capacity forbinding antibody is to make a very low-capacity resin such that a largervolume of resin slurry (20 uL for example) contains only enough capacityto bind 5 ug of antibody. An alternative approach is to reduce theeffective capacity of a resin by diluting the resin with an appropriatevolume of non-functionalized resin. For example, a protein G-sepharoseresin with a binding capacity of 20 ug/uL could be converted to a mixedresin with an effective binding capacity of 0.5 ug/uL by mixing 1 partof protein G-sepharose with 40 parts unfunctionalized sepharose. Inperforming such a resin dilution, in some embodiments, the diluent willbe a resin which is constructed from the same base material as theaffinity resin, has pore sizes small enough to exclude antibodies, andlacks any surface functionality which may interact with antibodies orthe chemical reagents used to prepare antibody conjugates.

In some aspects of the invention, antibodies are immobilized on a solidsupport by the step of applying an antibody-containing sample to a solidsupport. If desired, a washing step can be performed followingimmobilization to separate the immobilized antibodies from the cellculture supernatant or other components of the antibody-containingsamples.

Once the antibodies are immobilized on the solid support, a reductionstep is performed in order to fully reduce the reducible disulfide bondsof the immobilized antibodies and to generate reactive thiols. Theantibodies are reduced under conditions that are favorable to a completereduction of the reducible disulfide bonds. Typically, the antibodiesare reduced with an excess of reducing agent in order to ensure asubstantially complete reduction of the reducible disulfide bonds. Bythe phrase “fully reducing the reducible disulfide bonds of theantibody” it is meant that substantially all (e.g., greater than 70%,preferably greater than 80%, even more preferably greater than 85%, 90%,or 95%) of the antibodies in a sample are fully reduced as to theirreducible disulfide bonds. In other words, for a substantial amount ofthe antibodies in a sample, all of the antibodies' reducible disulfidebonds will be cleaved during the reduction step. For example, if theantibodies in a sample have 4 reducible disulfide bonds, after thereduction step, all 4 reducible disulfide bonds of a substantial amountof the antibodies will be cleaved to generate 8 reactive thiols. Thereduction is one that is selective for reducible disulfide bonds. By thephrase “selective for reducible disulfide bonds” it is meant that thereducible disulfide bonds are substantially the only bonds that arereduced. In some embodiments of the invention, the reducible disulfidebonds are the naturally occurring interchain disulfides of the antibody,the antibodies are reduced under conditions that are favorable to acomplete reduction of the naturally occurring interchain disulfides, andthe reduction is one that is selective for the naturally occurringinterchain disulfides. By the phrase “selective for interchaindisulfides” it is meant that interchain disulfides are selectivelyreduced. In other words, the interchain disulfides are substantially theonly bonds that are reduced. Because the antibodies are contacted withan excess of reducing agent and the reducing agent is selective for thereducible disulfide bonds, the generation of reactive thiols perantibody will generally be independent of the quantity of antibody inthe sample.

In one aspect, the reducing agent used in the reduction step is TCEP(tris(2-carboxyethyl)phosphine) and the TCEP is added at an excess for30 minutes at room temperature. For example, 250 uL of a 10 mM solutionof TCEP at pH 7.4 will readily reduce the interchain disulfides of 1 to100 ug of antibody in 30 minutes at room temperature. Other reducingagents and conditions, however, can be used. Examples of other reducingagents include DTT (dithiothreitol), mercaptoethanol andmercaptoethylamine. Examples of reaction conditions include temperaturesfrom 5° C. to 37° C. over a pH range of 5 to 8. Conjugation of theresulting antibody thiols and analysis by hydrophobic interaction orreveresed-phase chromatography (for examples, see FIGS. 1 and 3respectively) provides an indicator of the extent of disulfide reductionachieved under various reducing conditions. Following the reduction, awashing step can be performed in order to remove reducing agent and anyother components that may have nonspecifically attached to the solidsupport during the antibody capture step, for example, culture mediacomponents.

In some aspects of the present invention, although the samples will varywith respect to antibody quantity and antibody sequence, the majority ofantibodies will not vary substantially with respect to the number ofreducible disulfide bonds. For example, in some embodiments,substantially all of the antibody contained in the first and secondsample will have the same amount of reducible disulfide bonds. In somesuch embodiments, the reducible disulfide bonds will be interchaindisulfides. If the antibody in the first and second sample have 4interchain disulfide bonds (e.g., human IgG1), after reduction, thereduced immobilized antibodies in both samples will each have 8 reactivethiols. This level of reduction to 8 reactive thiols per antibody isindependent of the quantity of antibody in the samples due to the excessof reducing agent, the selectivity of the reduction step, and theuniform number of reducible bonds on each antibody. Similarly, if theantibody in the first and second sample have 5 interchain disulfidebonds, after reduction, the reduced immobilized antibodies in bothsamples will each have 10 reactive thiols. This level of reduction to 10reactive thiols per antibody is also independent of the quantity ofantibody due to the excess of reducing agent, the selectivity of thereduction step, and the uniform number of reducible disulfides on eachantibody. In some embodiments wherein a panel of murine antibodies isbeing screened, e.g., a panel of murine antibodies from hybridomas, amajority of the antibodies will be either one of the major murineisoforms IgG1 and IgG2a. Murine IgG1 and IgG2a isoforms both contain 5interchain disulfide bonds and after reduction, each antibody will have10 reactive thiols. Accordingly, a majority of the antibodies will havethe same amount of reducible disulfide bonds. Although the majority ofisoforms in these embodiments may be IgG1 and IgG2a, other isoforms maybe present as well. For example, murine IgG2b, murine IgG2c and murineIgG3 isoforms may be present as well. In instances where murine IgG2bisoforms are present, the reduction of these antibodies will generate 12reactive thiols as IgG2b isoforms have 6 interchain disulfide bonds. Insome embodiments, transgenic mice will be used for antibody productionand the mice can be genetically engineered to produce antibodies havinga certain isotype as well as antibodies having human IgG isotypes. Insome such embodiments, the mice can be engineered to only expressspecific isotypes. In some embodiments, the mice can be engineered toonly express only one isotype or one or two major isotypes.

Following the reduction step, the antibodies are loaded with the desiredchemical entity (in other words, conjugated to the desired chemicalentity). The selection of the chemical entities to be used depends inpart on the purpose of the assay. In some embodiments, the antibodieswill be screened for the purpose of selecting an antibody for use as anADC. In these embodiments, it is desirable for the antibodies to beconjugated to a drug. The antibodies can be conjugated directly to thedrug or indirectly via a linker. The drug and drug-linker can be anydrug or drug-linker that is effective for use as an ADC and that isthiol reactive. By the phrase “thiol reactive” it is meant that thechemical entity will react with a reactive thiol generated by reductionof a reducible disulfide bond and will form a covalent bond thereto.Thiol reactive drugs and drug-linkers include those drugs ordrug-linkers that aren't naturally thiol reactive but have beenderivatized with a thiol reactive agent to render them thiol reactive.The conditions used for conjugation are such that the drug willselectively react with a reactive thiol (either directly or through itslinker). Examples of thiol reactive groups that are highly selective forreactive thiols include, for example, maleimides, such asN-ethylmaleimide. Maleimides such as N-ethylmaleimide are considered tobe fairly specific to sulfhydryl groups, especially at pH values below7, where other groups are protonated. At pH 7, for example, the reactionwith simple thiols is about 1,000 fold faster than with thecorresponding amines Reactions of thiols with maleimides are very rapidat room temperature at pH 7.4, and 30 minutes is adequate to ensurecomplete reaction without risking conjugation of the maleimide to aminegroups. Accordingly, in some embodiments, the drug will be linked to theantibody via a maleimide group. Other reactive groups that are highlyselective for reactive thiols include, for example, iodoacetamides,vinyl sulfones, and aziridines.

In some embodiments, it will be desirable to fully load an antibody withdrug. In such embodiments, the desirable drug loading level will beequal to the number of reactive thiols per antibody. For example, insome such embodiments, the desired drug loading will be 10 drugs perantibody and the number of reactive thiols per antibody will be 10. Insome embodiments wherein a drug loading level which is equal to thenumber of reactive thiols is desired, the thiol reactive drug ordrug-linker will be provided in sufficient excess to the immobilizedantibodies in order to react with all of the available reactive thiols.Because the reaction is set up such that the drugs and drug-linkers tobe used in this step are thiol reactive and the conditions used areselective for conjugation to reactive thiols, the drug or drug-linkerselectively reacts with the reactive thiols (i.e., the drug and druglinkers do not substantially react with other sites on the antibody,including for example, other amino acids (e.g, lysine residues)).Because of this selectivity, it is possible to control the drug loadingand to design the experiment such that there will be a substantialuniform drug loading between samples. By the phrase “substantial uniformdrug loading between samples” it is meant that the average drug loadingbetween samples is substantially the same or, in other words, theaverage number of drug molecules per antibody in sample one will besubstantially the same as the average number of drug molecules perantibody in sample two. Some variance in drug loading can be expectedbut generally it will be within a variance of about 25%, preferablywithin a variance of 20% or even 10%. Accordingly, in some embodimentswhere a majority of the samples contain antibodies of the murine IgG1and IgG2a subtypes, if a thiol reactive drug or drug-linker is added tothe samples in sufficient excess to react with all of the availablereactive thiols, there will be an average of 10 drug molecules perantibody in the majority of samples. Because these samples have asubstantial uniform drug loading, once eluted, the concentration of thepurified ADCs can be determined by methods known in the art, e.g.,spectrophotometric methods, and their activities can be compared todetermine which antibodies are more or less active in an assay. Thiscomparison can be performed even if the antibodies to be compared areprovided at variable concentrations and in some embodiments, at unknownvariable concentrations. A comparison between antibodies provided atunknown variable concentration is aided by the ability to substantiallyuniformly load them with drug or drug-linker.

In some embodiments, it is not desirable to fully load an antibody withdrug or drug-linker. In some such embodiments, if a lower drug loadinglevel is desired, the immobilized antibodies can be reacted with both adrug or drug-linker and a thiol capping agent. The term “thiol cappingagent” is used herein to refer to an agent which selectively blocks areactive thiol. The drug or drug-linker and thiol capping agent will beprovided in a ratio of drug or drug-linker to thiol capping agent whichresults in the desired drug loading. Like the drug or drug-linker, thecapping agent will be highly selective for reactive thiols. Thiolreactive capping agents include those capping agents that aren'tnaturally thiol reactive but have been derivatized with a thiol reactiveagent to render them thiol reactive. Examples of thiol capping agentsthat can be used include maleimide capping agents such as, for example,N-ethyl maleimide. Other capping agents include, for example,iodoacetamide and iodoacetic acid. In some embodiments, both the drug ordrug-linker and thiol capping agent have the same type of thiol reactiveagent. For example, in some preferred embodiments, if the drug is to belinked to the antibody via a maleimide group, the capping agent willalso be linked to the antibody via the same type of maleimide group.This helps ensure that the relative reaction rates of the drug-linkerand capping agent are similar. Preferably, there will be no more thanabout a 100-fold different in the relative reaction rates, morepreferably no more than 10-fold, and even more preferably no more than5-fold difference in the relative reaction rates.

In one aspect, the ratio of capping agent and drug or drug-linker chosenwill be dependent on the desired level of drug loading. In someembodiments, the ratio of drug linker or drug to capping agent providedto the immobilized antibodies will be reflected in the ratio at whichthese reagents are conjugated to the antibodies. In embodiments when thedrug or drug-linker and capping agent are provided in molar excess, theratio of drug linker or drug to capping agent provided will be reflectedin the ratio at which these reagents are conjugated to the antibodies ifthe intrinisic thiol reaction rates of these two components are thesame. For example, if a reaction mixture to be used for conjugation hasa 1:1 mixture of drug linker or drug to capping agent, in embodimentswhere the instrinsic thiol reactive rates of the drug or drug-linker andcapping agent are the same and a majority of the samples compriseantibodies having 5 reducible disulfide bonds (e.g., antibodies of themurine IgG1 and IgG2a subtypes), following the reaction, there will bean average of 5 drug molecules per antibody and 5 capping agents perantibody in the majority of samples. It has been observed, however, bythe present inventors that the instrinsic thiol reaction rates of thedrug or drug-linker and capping agent are generally not the same, andconsequently, if the drug or drug-linker and capping agent are providedin excess, the ratio at which the drug or drug-linker and capping agentare provided to the samples comprising immobilized antibodies will notbe same ratio at which they are conjugated to the antibodies. In suchembodiments, the appropriate ratio of drug or drug-linker to cappingagent can be determined experimentally in order to achieve the desiredlevel of drug loading. Notably, as long as the drug or drug-linker andcapping agent are provided in excess (generally, an excess of at leastabout 3-fold) and the antibodies present in the samples havesubstantially the same number of reducible disulfide bonds, the ratiowill produce consistent (i.e., substantially uniform) levels of drugloading across samples regardless of quantity of antibody present on thesolid support.

In some preferred embodiments, the conjugation reaction of the antibodyto the drug or drug-linker and capping agent will be under kineticcontrol, not thermodynamic control. For example, under conditions inwhich the total moles of drug or drug-linker and capping agent providedto a sample containing the immobilized antibodies is equal to or lessthan the number of moles of reactive thiol in the sample, then the ratioof drug or drug-linker and capping agent provided to the samplecomprising the immobilized reduced antibodies will be reflected in theactual conjugation ratio of the antibodies to drug or drug-linker andcapping agent. Such a conjugation reaction can be said to be underthermodynamic control. For example, if 100 pmoles of a murine IgG1antibody (about 15 ug) were reduced with excess reducing agent toproduce 10 thiols per antibody, then 1 nmole of reactive thiol would bepresent. If a 1:1 mixture of drug or drug-linker to capping agent wereprepared such that the concentration of each was 0.5 mM and the totalconcentration was 1 mM, the addition of 1 uL of this solution to thereduced antibody would present a total of 1 nmole of drug or drug-linkerand capping agent. Assuming the drug or drug linker and capping agentand thiol reaction is a highly favorable one (for example, both of thedrug or drug-linker and capping agents are maleimido derivatives), theconjugate prepared by this procedure would have a 1:1 mixture of the twocompounds (the thiol-maleimide reaction is highly favorable andthermodynamics would effectively drive this reaction to completion).This would be true even if one of the compounds reacted at asubstantially faster rate than the other. In embodiments where it isdesirable for a plurality of sampes to be uniformly loaded, thisapproach would generally require that the quantity of antibody presentin each of the samples be known. Moreover, in embodiments where there isvariability in the amount of antibody between samples (such as a panelof antibodies from hybridomas), it would require a great deal of effortto tailor the quantity of drug or drug linker and capping agent to beadded to each sample in order to arrive at samples that aresubstantially uniformly loaded. In embodiments where the quantity ofantibody in the samples is unknown and/or there is variability betweensamples, it is generally preferable to manipulate the reaction so thatit is under kinetic control and accordingly, to provide theantibody-containing samples with an excess of total drug or drug-linkerand capping agent.

In some embodiments of the present invention, the chemical entities tobe conjugated to the reactive thiols of the reduced antibodies will beprovided in molar excess (molar excess as to the reactive thiols). Inthese embodiments, if the drug or drug-linker reacts more quickly with areactive thiol than the capping agent, the drug or drug-linker will bedisproportionately represented on the final conjugate. This is becausethe drug or drug linker and capping agent are effectively competing witheach other to react with a limiting number of available reactive thiols.If the drug or drug linker and capping agent are present at equalconcentrations in the reaction solution, they will only be conjugated atequal concentrations if their reaction rates are the same. By alteringthe composition of a reaction mixture such that the concentrations ofthe drug or drug linker and capping agent are not equal, the ratio atwhich they react with the available thiols can be controlled. Forexample, a slow-reacting drug or drug linker will be disproportionatelyunderrepresented on a conjugate prepared with a 1:1 mixture with afaster reacting capping agent. By changing the ratio to 2:1 in favor ofthe slower reacting drug or drug-linker, its representation on theconjugate will be increased. Thus, by the modulation of the ratio ofdrug or drug linker and capping agent provided to the samples comprisingthe immobilized reduced antibodies, a desired ratio of drug or druglinker and capping agent on the final conjugate can be achieved. Underconditions in which the total drug or drug linker and capping agent ispresent in excess relative to the available reactive thiols, theirdistribution on the final product will be independent of the startingthiol quantity. In this manner, a plurality of samples can besubstantially uniformly loaded even when the quantity of antibody in thesamples is unknown and/or there is variability between samples. In someembodiments, an appropriate volume of drug or drug-linker and cappingagnet is provided to the samples such than a molar excess of about 2fold (and ,even more preferably, a molar exces of 3-fold or more) oftotal reactants relative to total thiols is present. If the quantity ofantibody in the samples is unknown, each sample can be treated as if ithas the maximum amount of antibody. For many of the samples,significantly less than the maximum amount will be present and theexcess will be greater than 2 fold. This provision of excess reactantshaving a set ratio allows for variable quantities of antibodies across apanel to be treated with a large, fixed quantity of total drug or druglinker and capping agent to produce a panel of conjugates withcomparable loading of each drug or drug linker and capping agentpresent. The fact that equal treatment of samples results in comparablelevels of loading, regardless of the quantity of antibody initiallypresent in the sample, makes this method convenient for high-throughputapplications in which large numbers of antibodies are conjugated.

As discussed herein, although it is preferable that a majority of thesamples to be assayed do not vary with respect to the number ofreducible disulfide bonds present on the antibodies contained therein,in some embodiments, there will be some variation. In some embodiments,despite the variation, the samples will be treated with the same ratioof chemical entities. When interpreting the data, the skilled artisanwill recognize that a certain subset of the samples differed in theamount of reducible disulfide bonds. If desired, the skilled artisan candetermine the antibody isotype prior to or post conjugation to aid indata interpretation.

In some embodiments, prior to the conjugation step, standard methods canbe used to determine the antibody isotype in each of the samples andtherefore, the number of reducible disulfide bonds per antibody in eachof the samples. In some such embodiments, samples that containantibodies having the same number of reducible disulfide bonds will becontacted with a reaction mixture having one ratio of drug or druglinker to capping agent to arrive at a desired drug loading and samplesthat contain antibodies having a differing number of reducible disulfidebonds will be contacted with a reaction mixture having a different ratioof drug or drug linker to capping agent to arrive at that same desireddrug loading. For example, in some embodiments, if the desired averagedrug loading is 4, samples that contain antibodies of murine IgG1 andIgG2a (10 reactive thiols per antibody when fully reduced) will all becontacted with a reaction mixture having a ratio of drug or drug linkerto capping agent to arrive at an average drug loading of 4 and averagecapping agent loading of 6. Samples that contain antibodies of murineIgG2b (12 reactive thiols per antibody when fully reduced) will becontacted with a reaction mixture having a different ratio of drug ordrug linker to capping agent (e.g., a higher fraction of capping agent)to arrive at the same average drug loading of 4. In other embodiments,although there may be variation between isotypes and number ofinterchain disulfides, it will be accepted that there will be somevariation in loading and all of the samples will receive the same ratioof drug or drug linker to capping agent.

In some embodiments, prior to the conjugation step and following thereduction step, there will be a partial reoxidation step. For example,in some embodiments, the reducible disulfide bonds will consist ofnaturally occurring interchain disulfide bonds as well as disulfidebonds formed from introduced sulfhydryl groups. In some of theseembodiments, it will be desirable to conjugate the selected chemicalentities to the introduced sulfhydryls but not to the sulfhydryl groupsof the naturally occurring interchain disulfide bonds. In theseembodiments, following the complete reduction of the reducible disulfidebonds, there can be a partial reoxidation step to reoxidize thenaturally occurring interchain disulfide bonds leaving the introducedsulfhydryls available for binding to the desired chemical entities.Reoxidation of the native disulfides can be achieved, for example, bytreatment of the reduced antibodies with a large molar excess ofdehydroascorbic acid at pH 6.5, with the reaction allowed to proceed for1 hour at room temperature.

In any of the embodiments described herein, instead of, or in additionto the capping agent, a detection agent is provided for conjugation. Thedetection agents can be, for example, primary labels or secondarylabels. In some embodiments, the detection agent will be one that isdetected directly. In other embodiments, the detection agent will be onethat is detected indirectly. In some embodiments, the detection agentwill be, for example, any thiol reactive label that can be used forantibody quantiation and/or as a reporter for a binding assay or anyother desirable assay. Thiol reactive labels include those labels thataren't naturally thiol reactive but have been derivatized with a thiolreactive agent to render them thiol reactive. In some embodiments, thesame type of thiol reactive agent will be used to link the variouschemical entities (detection agent and/or drug or drug-linker and/orcapping agent) to the antibody. In some embodiments, the detection agentwill be a radioactive compound, a chemiluminescent agent, a fluorescentagent, or a chromogen. In some embodiments, the detection agent will bea fluorescent molecule such as a fluorophore. In some embodiments, thedetection agent will be biotin. In one aspect, the detection agent willbe a fluorophore and the fluorophore will be derivatized with amaleimide group in order to make it thiol reactive. The teachingsdescribed herein can be used to assess the preferred loading level of aselect detection agent. In some embodiments, a fluorophore is used asthe detection agent and the fluorophore is loaded at an average loadingof about 2.5 to about 3 fluorophores per antibody. Examples 3 and 4provide exemplary descriptions of how to tailor the ratio of chemicalentities in order to achieve a desired drug and/or fluorophore loadinglevel.

The present invention encompasses embodiments wheren the antibodies arescreened not for the purpose of selecting an antibody for use as an ADCbut for the purpose of selecting an antibody for use as an unconjugatedantibody. In these embodiments, immobilized antibodies will be contactedwith a detection agent and capping agent at a selected ratio and therewill be no use of drug or drug-linker. Using the teachings describedherein, including the teachings of examples 3 and 4, the appropriateratio of detection agent to capping agent can be determined.

After contacting the reduced antibodies with the appropriate amount andtype of chemical entities (selection of the chemical entities will bedependent, for example, on whether it is desired to screen antibodies asunconjugated antibodies or ADCs; whether it is desired to have a fulldrug loading or partial drug loading; and whether it is desired toinclude a detection agent in the mix) and allowing sufficient time forcompletion of the reaction (e.g., 30 minutes for maleimide-containingchemical entities), it is desirable to perform a washing step in orderto remove any unreacted materials. Subsequently, the immobilizedantibody conjugates can be eluted from the solid support to provideantibody conjugate compositions. Methods of eluting proteins from solidsupports are known in the art and the skilled practitioner will be ableto select an appropriate buffer for elution. For example, inembodiments, where the solid support comprises protein A or protein Gresin, the antibody conjugates can be eluted with standard low pHbuffers for elution from protein A or protein G columns.

In some embodiments of the invention, the methods described herein formaking antibody conjugates will result in a plurality of antibody drugconjugate compositions having substantially uniform drug loading (theskilled artisan will understand that there may be some outliersdepending on the uniformity of number of reducible disulfide bondsacross samples). In these embodiments, because of the substantiallyuniform drug loading between samples, the relative characteristics ofantibodies in a first and second sample can be compared. This comparisoncan be performed even though the antibodies to be compared were providedat variable concentrations and, in some embodiments, at unknown variableconcentrations. A comparison between the antibodies of unknown andvariable concentration is made easier with the ability to substantiallyuniformly load them with drug or drug-linker.

Methods for determining drug loading are known in the art. One methodthat is used herein is high-performance liquid chromatography on apolystyrene divinylbenzene copolymer, e.g., a reversed-phase PLRP™column. This denaturing technique can cleanly separate the variouslyloaded light chain and heavy chain species. Hydrophobic interactionchromatography (HIC) can also be used as an analytical method used todetermine isomeric mixtures from resultant conjugates. The drug loadinglevel can be determined based on a ratio of absorbances, e.g., at 250 nmand 280 nm. See, for example, U.S. Publication No. 20090010945.

In some embodiments, following elution of the antibody conjugates,activity assays and/or other assays will be performed in order tocharacterize the antibody conjugates. In some embodiments, cell binding,affinity, and/or cytoxicity assays will be performed. Many methods ofdetermining whether an ADC binds a target of interest or exerts acytotoxic effect on a cell are known to those of skill in the art, andcan be used in the present methods. For example, cell viability assayscan be used to determine the cytotoxic effect of an ADC on a cell. See,for example, U.S. Pat. Nos. 7,659,241 and 7,498,298, each of which isincorporated herein in its entirety and for all purposes, for exemplarycell binding and cytotoxicity assays.

In some embodiments, following elution of the antibody conjugates, itwill be desirable to determine the quantity of antibody or antibodyconjugate in the antibody conjugate compositions. In some embodiments,it will be desirable to determine the actual quantity of antibody orantibody conjugate in a sample. In other embodiments, it will besufficient to determine the relative quantity of antibody or antibodyconjugate in a plurality of samples. For example, it may be sufficientto know that sample 1 has more antibody than sample 2 which has moreantibody than sample 3, and so forth. Many methods for determiningprotein quantity are known in the art and can be used herein. In someembodiments, an absorbance assay will be used to determine antibodyconcentration. In embodiments where a fluorophore is part of theantibody conjugate, antibody concentration can be determined using afluorescence assay. In embodiments where fluorescence is used forprotein quantitation, a standard may be necessary to convert the rawfluorescence values into a concentration. Methods of using fluorescenceand generating standard curves to determine protein concentration areknown in the art. In one example, approximately 200 μg of a standardantibody will be conjugated during the conjugation step after beingspiked into blank media. After elution, the concentration of thisstandard will be determined by conventional methods, e.g., aconventional A280 absorbance assay, and a standard curve prepared by adilution series will be assayed for fluorescence alongside the conjugatesamples. Alternatively, a liquid-handling robot can be used to normalizeplates thereby eliminating the need for serial dilutions.

In some embodiments, the results of a cytotoxicity assay and knowledgeof the relative or actual antibody concentration in the antibodyconjugate compositions will be used to identify antibodies with desiredcharacteristics. The methods described herein for making antibodyconjugates allow for comparisons to be made between a plurality ofantibodies of varying concentration and, in some embodiments, unknownquantity. The methods described herein for making antibody conjugatesallow for a selection of antibodies with desirable characteristics whenstarting with, for example, a panel of antibodies resulting from ahybridoma fusion. In some preferred embodiments, it is the substantialuniform drug loading between samples that allows for relevantcomparisons to be made between samples. Failure to ensure substantiallyuniform loading levels, could, for example, lead to erroneous resultsfrom a screen of a panel of antibodies for use as ADCs. This is becauseit would not be known if an ADC sample exhibited greater cytotoxicitybecause of the characteristics of the antibody as an ADC or because thesample contains more drug per antibody. For example, an antibodyconjugate composion comprising antibody “A” and having an average drugloading of 4 would typically be expected to exhibit more cytotoxicitythan an antibody conjugate composition comprising antibody “B” andhaving an average drug loading of 1. This greater cytotoxicity would notbe an indicator of the relative characteristics of antibodies A and B asADCs, but simply an indicator of the greater drug loading on antibody A.If both antibody conjugate compositions had an average drug loading ofabout 4, if one showed greater cytotoxicity, it could be attributed tothe antibody and not simply the drug loading. Similarly, the ability todetermine the actual or relative quantity of antibody or antibodyconjugate in the samples also allows for relevant comparisons to be madebetween samples. Without knowledge of actual or relative quantity ofantibody or antibody conjugate in the sample, it would not be known ifan ADC exhibited greater cytotoxicity because of the particular antibodyor simply because there is more antibody or ADC in the sample.

In addition to providing methods for making antibody conjugates for usein antibody screening assays and antibody conjugates produced by theclaimed methods, the present invention provides antibodies and/orantibody conjugates (e.g., antibody drug conjugates) for therapeutic usewherein the antibody was selected using the methods described herein.

As previously discussed, the drug or drug-linker used in the presentmethods can be any drug or drug-linker that is effective for use as anADC and that is thiol reactive. The drug can be any cytotoxic,cytostatic or immunosuppressive drug. Methods of selecting drug anddrug-linker for use as ADCs are known in the art. See, for example, WO2004010957, WO 2007/038658, U.S. Pat. No. 6,214,345, U.S. Pat.No.7,498,298, and U.S. Publication No. 2006/0024317, each of which isincorporated herein by reference in its entirety and for all purposes.

Useful classes of cytotoxic or immunosuppressive agents include, forexample, antitubulin agents (e.g., auristatins, maytansinoids, vincaalkaloids), topoisomerase inhibitors (e.g., camptothecins), DNA minorgroove binders (e.g., calicheamicins, duocarmycins, enediynes,lexitropsins, chloromethylbenzindolines), DNA replication inhibitors(e.g., anthracyclines), alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), protein kinase inhibitors, cytotoxicenzymes, and protein toxins.

In some embodiments, suitable cytotoxic agents include, for example,antibiotics, antifolates, antimetabolites, chemotherapy sensitizers,etoposides, fluorinated pyrimidines, ionophores, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, radiationsensitizers, steroids, puromycins, doxorubicins, and cryptophysins.

Individual cytotoxic or immunosuppressive agents include, for example,an androgen, anthramycin (AMC), asparaginase, 5-azacytidine,azathioprine, bleomycin, busulfan, buthionine sulfoximine, gammacalicheamicin, N-acetyl gamma dimethyl hydrazide calicheamicin,camptothecin, carboplatin, carmustine (BSNU), CC-1065, cemadotin,chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine,cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin(formerly actinomycin), daunorubicin, decarbazine, discodermolide,docetaxel, doxorubicin, morpholino-doxorubicin,cyanomorpholino-doxorubicin, echinomycin, eleutherobin, epothilone A andB, etoposide, estramustine, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin,ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine,melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C,mitoxantrone, netropsin, nitroimidazole, paclitaxel, palytoxin,plicamycin, procarbizine, rhizoxin, streptozotocin, tenoposide,6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine,vinorelbine, VP-16 and VM-26.

In some embodiments, the drug is an anti-tubulin agent. Examples ofanti-tubulin agents include, but are not limited to, taxanes (e.g.,Taxol® (paclitaxel), Taxotere® (docetaxel)), and vinca alkyloids (e.g.,vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulinagents include, for example, baccatin derivatives, taxane analogs (e.g.,epothilone A and B), nocodazole, colchicine and colcimid, estramustine,cryptophysins, cemadotin, combretastatins, discodermolide, andeleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 or DM-4 (ImmunoGen, Inc.; see alsoChari et al., 1992, Cancer Res. 52:127-131).

In some embodiments, the drug is an auristatin, another group ofanti-tubulin agents. Auristatins include, but are not limited to,auristatin E and derivatives thereof. AFP, AEB, AEVB, MMAF, and MMAE areexamples of auristatins that can be used herein. The synthesis andstructure of auristatins are described in U.S. Patent ApplicationPublication Nos. 2003-0083263, 2005-0238649 and 2005-0009751;International Patent Publication No. WO 04/010957, International PatentPublication No. WO 02/088172, and U.S. Pat. Nos. 7,498,298, 6,323,315;6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483;5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024;5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and4,486,414, each of which is incorporated herein by reference in itsentirety and for all purposes.

The linker part of a drug-linker is a compound that can be used to linkthe antibody to the drug. The linker can comprise more than one chemicalmoiety. In some embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker releases the drug unit fromthe antibody in the intracellular environment. In some embodiments, thelinker is a peptidyl linker (e.g. a linker that comprises two or moreamino acids) that is cleaved by an intracellular peptidase or proteaseenzyme, including, but not limited to, a lysosomal or endosomalprotease. Cleaving agents can include cathepsins B and D and plasmin,all of which are known to hydrolyze dipeptide drug derivatives resultingin the release of active drug inside target cells (see, e.g., Dubowchikand Walker, 1999, Pharm. Therapeutics 83:67-123). In some embodiments,the peptidyl linker cleavable by an intracellular protease comprises aVal-Cit dipeptide or a Phe-Lys dipeptide (see, e.g., U.S. Pat. No.7,659,241, incorporated by reference herein in its entirety and for allpurposes). In yet other embodiments, the linker is not cleavable and thedrug is released by antibody degradation.

In some embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values and/or cleavable underreducing conditions (e.g., a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

Exemplary linkers that can be used with the present methods aredescribed in WO 2004010957, WO 2007/038658, U.S. Pat. Nos. 6,214,345,7,659,241, 7,498,298 and U.S. Publication No. 2006/0024317, each ofwhich is incorporated herein by reference in its entirety and for allpurposes.

In some exemplary embodiments of the present invention, the drug-linkeris of Formula I or Formula II wherein Val-Cit refers to the dipeptidevaline-citrullline.

Proteins

The methods described herein for making antibody conjugates can also beused to make fusion proteins for use in fusion protein screening assays.The term “fusion protein” is used herein to refer to binding domain-Igfusions, wherein the binding domain may be, for example, a ligand, anextracellular domain of a receptor, a peptide, a non-naturally occurringpeptide or the like with the proviso that the binding domain does notinclude a variable domain of an antibody. Like the antibodies describedherein, the Ig portion of the fusion protein must comprise at least onereducible disulfide bond, and a domain capable of binding to a solidphase. In one aspect, the Ig domain will be the Fc region of anantibody. Examples of domain-Ig fusion proteins include etanercept whichis a fusion protein of sTNFRII with the Fc region (U.S. Pat. No.5,605,690), alefacept which is a fusion protein of LFA-3 expressed onantigen presenting cells with the Fc region (U.S. Pat. No. 5,914,111), afusion protein of Cytotoxic T Lymphocyte-associated antigen-4 (CTLA-4)with the Fc region (J. Exp. Med. 181:1869 (1995)), a fusion protein ofinterleukin 15 with the Fc region (J. Immunol. 160:5742 (1998)), afusion protein of factor VII with the Fc region (Proc. Natl. Acad. Sci.USA 98:12180 (2001)), a fusion protein of interleukin 10 with the Fcregion (J. Immunol. 154:5590 (1995)), a fusion protein of interleukin 2with the Fc region (J. Immunol. 146:915 (1991)), a fusion protein ofCD40 with the Fc region (Surgery 132:149 (2002)), a fusion protein ofFlt-3 (fins-like tyrosine kinase) with the antibody Fc region (Acta.Haemato. 95:218 (1996)), a fusion protein of OX40 with the antibody Fcregion (J. Leu. Biol. 72:522 (2002)), and fusion proteins with other CDmolecules (e.g., CD2, CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-I),CD137), adhesion molecules (e.g., ALCAM (activated leukocyte celladhesion molecule), cadherins, ICAM (intercellular adhesion molecule)-1,ICAM-2, ICAM-3) cytokine receptors (e.g., interleukin-4R,interleukin-5R, interleukin-6R, interleukin-9R, interleukin-10R,interleukin-12R, interleukin-13Ralpha1, interleukin-13Ralpha2,interleukin-15R, interleukin-21Ralpha), chemokines, cell death-inducingsignal molecules (e.g., B7-H1, DR6 (Death receptor 6), PD-1 (Programmeddeath-1), TRAIL R1), costimulating molecules (e.g., B7-1, B7-2, B7-H2,ICOS (inducible co-stimulator)), growth factors (e.g., ErbB2, ErbB3,ErbB4, HGFR), differentiation-inducing factors (e.g., B7-H3), activatingfactors (e.g., NKG2D), and signal transfer molecules (e.g., gpl30),BCMA, and TACI.

All of the steps described herein can easily be adapted to embodimentswherein the starting material is not antibody but fusion protein. Forexample, in some embodiments, fusion protein-containing samples would beprovided in lieu of antibody-containing samples. The fusion proteinsamples would vary with respect to quantity and sequence. In preferredembodiments, substantially all of the fusion protein present in a singlesample would be of the same sequence. “Substantially all of the fusionprotein present in a single sample is of the same sequence” reflects thepreference that a single sample contain one fusion protein with therecognition that there may be a minor amount (e.g., up to 20%,preferably less than 15%, less than 10%, less than 5%, less than 4%, orless than 3%) of contamination with another fusion protein.

As with the antibodies, the methods would not require a purificationstep prior to fusion protein immobilization. In some aspects, the fusionprotein provided in the fusion protein-containing sample is notpurified. As with the antibodies, in some embodiments, unpurifed cellculture supernatatant is provided as the fusion protein-containingsample. Methods of generating fusion proteins in cell culture are knownin the art and not discussed herein. In some embodiments, fusion proteinin the fusion protein-containing samples was grown in IgG depletedculture medium, and, in particular, culture medium depleted of bovineIgG. As with the antibodies, the present methods can be performed withsamples that contain very small amounts (e.g., 1 to 500 μg) of fusionprotein. In some embodiments, there will be from 1 μg to 100 μg, from 1μg to 50 μg, from 1 μg to 20 μg, from 5 μg to 100 μg, from 5 μg to 50μg, from 5 μg to 20 μg of fusion protein present in a single sample.

The present methods comprise a step of immobilizing the fusion proteinon a solid support to provide immobilized fusion proteins. In someembodiments, the solid support has the capacity to bind more fusionprotein than the amount present in the fusion protein-containing sampleor the amount of bound fusion protein is less than the capacity of thesolid support. In other embodiments, the solid support will have reducedbinding capacity.

Once the fusion proteins are immobilized on the solid support, areduction step is performed in order to fully reduce the reducibledisulfide bonds of the immobilized fusion protein and to generatereactive thiols. Following the reduction step, the fusion proteins areloaded with the desired chemical entity (in other words, conjugated tothe desired chemical entity). Again, the selection of the chemicalentities to be used depends in part of the purpose of the assay. In someembodiments of the present invention, the fusion proteins will bescreened for the purpose of selecting fusion protein for use as a fusionprotein drug conjugates. In these embodiments, it is desirable for thefusion proteins to be conjugated to a drug. The fusion proteins can beconjugated directly to the drug or indirectly via a linker. The drug anddrug-linker can be any drug or drug-linker described herein. As with theantibodies, the fusion proteins can be contacted with a reaction mixturecomprising drug, capping agent and optionally a detection agent. As withthe antibodies, the present invention encompasses embodiments wheren thefusion proteins are screened not for the purpose of selecting a fusionprotein for use as an fusion protein drug conjugate but for the purposeof selecting a fusion protein for use as an unconjugated fusion protein.In these embodiments, the conjugation reaction mixture will not includea drug or drug linker but instead a mixture of detection agent andcapping agent. As with the antibody conjugates, in some embodiments, themethods described herein for making fusion protein conjugates willresult in a plurality of fusion protein conjugate compositions withsubstantial uniform loading between samples. Following elution of thefusion proteins, activity assays and/or other assays can be performed inorder to characterize the fusion proteins. The results of the assays andknowledge of the relative or actual protein concentration in the fusionprotein conjugate compositions can be used to identify fusion proteinsthat have desired properties either as unconjugated fusion proteins oras fusion protein drug conjugates.

Using the methods described herein, antibodies that perform well asunconjugated antibodies and fusion proteins that perform well asunconjugated fusion proteins can be identified and can be selected forfurther development. In some embodiments, antibodies or fusion proteinsidentified by the present methods will be formulated for therapeuticand/or non-therapeutic applications. Similarly, antibodies or fusionproteins identified as those with desired activites as drug conjugatescan also be selected for further development. In some embodiments, suchantibodies or fusion proteins will be conjugated to the desired drug ordrug-linker using known methods and will be formulated for therapeuticand/or non-therapeutic applications. In some embodiments, theantibodies, antibody drug conjugates, fusion proteins, and fusionprotein conjugates will be formulated as pharmaceutical compositions andwill comprise a therapeutically or prophylactically effective amount ofthe antibody, antibody-drug conjugate, fusion protein, or fusion proteinconjugate and one or more pharmaceutically compatible (acceptable)ingredients. For example, a pharmaceutical or non-pharmaceuticalcomposition typically includes one or more carriers (e.g., sterileliquids, such as water and oils). Water is a more typical carrier whenthe pharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable excipients include, for example, amino acids, starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene glycol, water, ethanol, and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin. Such compositions willtypically contain a therapeutically effective amount of the protein,typically in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulations correspond to the mode of administration.

Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. When necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. When thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. When the pharmaceutical is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

EXAMPLES Example 1 Reduction of Antibodies in Solution and by SolidPhase

It is well recognized that under conditions in which antibodies retaintheir native folded structure, TCEP readily reduces the interchaindisulfides without reducing the intrachain disulfides of theimmunoglobulin domains, which are inaccessible to water-solublereagents. When an antibody is bound to protein G affinity media, thisselectivity for the interchain disulfides remains unchanged. This isillustrated in FIG. 1. This figure shows chromatograms made by reducinga protein G-immobilized murine antibody with 10 mM TCEP, followed byconjugation with an excess of mc-MMAF. These chromatograms are overlaidwith chromatograms of the same antibody reduced with TCEP byconventional solution chemistry and reacted with mc-MMAF. The comparableresults between the standard solution method and the solid phase methodindicate that the reactivity of the antibody is not significantlychanged upon binding to protein G affinity media. This feature allows alarge panel of antibodies to all be reduced to the same number ofreactive thiols without regard to the quantity of each antibody present,by using a quantity of TCEP that is in excess to the number of reducibledisulfides in the most abundant antibody. In the absence of anyknowledge of how much antibody may be present, the most theoreticallyabundant antibody may be defined as the capacity of the affinity resin(ug antibody per uL resin) times the volume of the resin bed (uL).

Example 2 Tailoring the Ratio of Drug to Capping Agent in the DrugConjugation Reaction Mixture for a Desirable Drug Loading

FIG. 2 illustrates the degree of loading of the maleimido drug mcMMAFwhen added as a mixture with N-ethyl maleimide (NEM) to a murine IgG1immobilized on protein G and fully reduced with excess TCEP. The figureillustrates the slightly lower reactivity of mcMMAF relative to NEM,such that, in this example, if a conjugate with an average mcMMAF molefraction of 0.4 is desired (a drug loading of 4), the mole fraction ofmcMMAF in the maleimide mixture must be 0.53. The loading of mcMMAF oneach conjugate was determined by reversed-phase chromatography with aPLRP-S column, which effectively separates the heavy and light chains onthe basis of their drug loading; the hydrophobicity of mcMMAF results inlater retention times for species with increasing degree of mcMMAFconjugation (FIG. 3). A mixture of mcMMAF and NEM was prepared at thisratio and applied to a small panel of murine antibodies to assess thegenerality of this ratio across different IgG isotypes. As shown in thetable below, murine IgG1's and IgG2a's, both of which possess 5interchain disulfides, gave mcMMAF drug loading levels between 3.9 and4.2 as determined by PLRP-S chromatography. A murine IgG2b, whichpossesses 6 interchain disulfides, gave a correspondingly greateraverage mcMMAF loading as a result of the greater number of reactivethiols per antibody which result from complete reduction. This resultillustrates the importance of tailoring the maleimide mixture accordingto the number of reducible antibody disulfides if a specific loadinglevel is desired.

Isotype mIgG1 mIgG2a mIgG2b Drugs/Ab 3.9 4.0 3.9 4.2 4.2 4.1 3.9 5.3

Example 3 Method for Determining Exemplary Fluorophore Loading Level andfor Preparing a Standard for Determining Fluorophore Loading in AntibodyConjugates

Mixed conjugates can be prepared with both drug and a fluorophorepresent on the conjugate in a controllable manner The presence of afluorophore can enable more sensitive quantitation of the conjugatesresulting from a large panel of antibodies or as a reporter group forbinding assays or other assays performed on the panel. Alexa Fluor® 647maleimide can be included in a mixture of maleimides, along with mcMMAFand NEM, to create a panel of antibody conjugates with a desired averageloading for Alexa Fluor® 647 and mcMMAF. To assess a targeted loadinglevel of AlexaFluor 647, a series of murine IgG1 conjugates was preparedusing a binary mixture of AlexaFluor 647 maleimide and mcMMAF. Theaverage loading of mcMMAF on these conjugates was determined by PLRP-Schromatography, and the loading of Alexa Fluor® 647 was calculated as(10-mcMMAF loading), as the total conjugation sites on fully reducedmurine IgG1 is 10. The fluorescence output of these conjugates was thendetermined using a fluorescence plate reader, and plotted as a functionof Alexa Fluor® 647 loading (FIG. 4). FIG. 4 illustrates thatfluorescence rapidly increases with increasing loading level up to amaximum value corresponding to about 2.5 to about 3 fluorophores perantibody, then steadily declines with further fluorophore loading. Thisdecrease in fluorescence output with increasing fluorophore loading ispresumably due to self-quenching which arises from the close spatialproximity of the fluorophores when conjugated to the reduced disulfidesof an antibody. Based on this result, a fluorophore loading ofapproximately 3 per antibody was selected. At this loading level notonly would the fluorescence output be maximal (resulting in greatestsensitivity in fluorescent assays), but also the variation influorescence as a function of fluorophore loading will be minimal. Thiswill ensure that small variations in fluorophore loading across anantibody panel will not result in large differences in fluorescentoutput, an important point if fluorescence is to be used to quantify theconjugate concentrations.

The ratio of the absorbance at 650 nm to 280 nm was also determined foreach of the fluorophore-drug conjugates described above. These ratiosare shown in FIG. 5, plotted against the fluorophores per antibody data.In the region of 2.5 to 3 fluorophores per antibody, the change in the650 nm/280 nm absorbance ratio is linear with the change in loadinglevel, and the equation of this line can be used to determine thefluorophore loading in mixed AF647-mcMMAF antibody conjugates from themeasured absorbance values.

Example 4 Exemplary Method for Tailoring the Ratio of Chemical Entitiesin Order to Achieve a Desired Drug Loading Level

As described in example 3, an exemplary number of fluorophores perantibody is about 3. Assuming that the antibody-containing samples areof the murine IgG1 and IgG2a isotypes and the desired loading level forflurophores is 3, a drug loading level of 7 could be achieved bypreparing the appropriate mixture of AF647 maleimide and mcMMAF.However, a lower level of drug loading may be achieved by including acapping reagent such as N-ethyl maleimide (NEM). Thus, a ternary mixtureof AF647, mcMMAF, and NEM could be prepared in an appropriate ratio toachieve any desired level of AF647 and mcMMAF loading (provided that thesum of the two is no greater than 10 for a murine IgG1 or IgG2a). Todetermine the correct mixture of these three reagents necessary toachieve a desired loading level, their relative reactivities weredetermined. This was done by preparing 1:1 mixtures of mcMMAF:NEM andAF647:NEM and reacting these mixtures with a fully reduced murine IgG1immobilized on Protein G. The level of fluorophore loading in theresulting AF647 conjugate was determined from its 650 nm/280 nmabsorbance ratio by reference to FIG. 5, while the mcMMAF loading in theresulting drug conjugate was determined by PLRP chromatography. Thesedata are shown in the table below; the mole fraction on antibody is theloading of each reagent (AF647 or mcMMAF) divided by 10, the totalnumber of maleimides which conjugate to the reduced murine IgG1; the NEMmole fraction is 1 minus the reagent mole fraction; and the relativereactivity is the ratio of the reagent mole fraction to the NEM molefraction. In this analysis, NEM is assigned a relative reactivity valueof 1.

NEM mole Mole fraction fraction on Relative 1:1 mix Loading on antibodyantibody Reactivity AF647:NEM 2.88 0.288 0.712 0.404 mcMMAF:NEM 3.750.375 0.625 0.6

To convert these relative reactivity values into an appropriate ratio ofmaleimides to use in the ternary mixture, it is first necessary todefine the desired loading levels of each reagent on the finalconjugated antibody. For this example, a target loading of 4.5 mcMMAF, 3AF647, and 2.5 NEM will be used, again assuming that the antibodyconjugate will have 10 available thiols when reduced. This correspondsto a conjugated mole fraction of 0.45, 0.3, and 0.25, respectively. Thenecessary calculations are then summarized in the table below.

Conjugated required mole Mole Fraction Relative Reactivity Mole Fractionfraction in Reagent Target (see table above) Relative Reactivitymaleimide mix mcMMAF 0.45 0.6 0.75 0.43 AF647 0.3 0.404 0.74 0.43 NEM0.25 1 0.25 0.14

Briefly, the target value for the conjugated mole fraction of eachreagent is divided by its relative reactivity factor which wasdetermined above using 1:1 mixes of the different reagents. This valueis then converted to a required mole fraction in the maleimide mixtureby dividing the value by the sum of the values for all reagents. Forexample, for mcMMAF, 0.45/0.6=0.75; 0.75/(0.75+0.74+0.25)=0.43. In thismanner, a mixture of mcMMAF, AF647, and NEM in a ratio of 0.43:0.43:0.14would be predicted to yield antibody conjugates with an average loading4.5, 3, and 2.5 for the three reagents, respectively. In like manner,different ratios of the reagents could be calculated to achievedifferent loading levels on the conjugate, or ratios for other reagentscould be calculated once their relative reactivities had beendetermined.

Example 5 Demonstration of the Consistency of Drug Loading AcrossSamples

A solution of mcMMAF, AF647 maleimide, and NEM was prepared in a ratioof 0.43:0.43:0.14 and used to conjugate a panel of antibodies by thepresent methods. These antibodies had been generated from 1.5 mL ofbovine IgG-depleted hybridoma culture media, using newly fusedhybridomas resulting from a murine immunization campaign. One 96-wellplate of samples was subjected to analysis to determine the drug andfluorophore loading consistency of the resulting mixed conjugates. Thefluorophore loading was determined by the 650 nm/280 nm absorbance ratioof each sample, measured in an absorbance plate reader, by reference tothe linear relationship shown in FIG. 5. The resulting data are shown inFIG. 6, plotted against the quantity of conjugate that each sampleyielded; the data are shown only for those samples which yielded atleast 2.5 μg of conjugate, as lower quantities than this did not produce280 nm absorbance values significantly above the baseline. There were 65samples on the plate which met this 2.5 μg threshold and are plotted inFIG. 6. As can be seen in the figure, the loading is scattered between 2and 4 fluorophores per antibody, with a calculated mean of 3.26 and acoefficient of variation of 10.2%. The mean loading of 3.26 differs byless than 10% from the targeted loading of 3. Importantly, the range ofobserved fluorophore loading levels fell within the region of thefluorescence vs loading curve (FIG. 4) where the fluorescence does notchange greatly, due to the self-quenching phenomenon. In other words,the difference in observed fluorescence between antibodies with 2, 3, or4 fluorophores per antibody is expected to be less than 20%. The mcMMAFloading was determined by PLRP chromatography; a sample PLRPchromatogram of an mcMMAF-AF647-NEM antibody conjugate is shown in FIG.7. This figure is an overlay of two analytical wavelengths, 280 nm todetect all of the peaks containing protein, and 620 nm to detect thosepeaks containing at least one Alexa Fluor® 647. As can be seen in thisfigure, the light chain with NEM (2.2 minutes) is slightly resolved fromthe light chain with Alexa Fluor® 647 (2.5 minutes), but both are wellresolved from the light chain with mcMMAF (3.8 minutes), illustratingthat the PLRP column separates species well on the basis of mcMMAFloading but not AF647 loading. Since the light chain contains only onecysteine which is reduced by the TCEP treatment, these are the onlylight chain species present, and the NEM and mcMMAF peaks do not haveabsorbance at 620 nm as they contain no AF647. The heavy chain peakcluster is more complicated due to the fact that with 4 availablethiols, each peak is not a single species. For example, the peakcorresponding to heavy chain with 2 copies of mcMMAF (7.7 minutes) is acollection of heavy chain species which also contain 2 AF647, 2 NEM, or1 AF647 and 1 NEM; these various species are not separated by the PLRPcolumn This feature of the separation permits these data to be used toassess strictly the mcMMAF loading without being affected by thepresence of AF647 or NEM. Using this method, the mcMMAF loading levelswere determined for 34 samples from the plate of hybridoma supernatants,and are plotted in FIG. 8 against the quantity of conjugate that eachsample yielded. As can be seen in the figure, the loading is scatteredbetween 4 and 6 copies of mcMMAF per antibody, with a calculated mean of4.51 and a coefficient of variation of 7.75%. The mean loading of 4.51is exactly at the targeted level of 4.5. As can be seen in the figure,there are 3 outliers with loading levels greater than 5; the PLRPchromatograms from these samples contain a heavy chain species with 5copies of mcMMAF, indicating the presence of an additional reducibledisulfide on the heavy chain of these antibodies (see FIG. 9 as anexample), suggesting that these antibodies are of the murine IgG2bisotype. Thus, the higher loading observed in these samples is not dueto disproportionate loading of mcMMAF compared to the other antibodies,but rather that these antibodies have 20% more available thiols (12rather than 10) and therefore would be expected to be loaded with eachreagent at levels 20% higher. If these three are excluded from theanalysis and only those antibodies with 10 available thiols areconsidered, the mean mcMMAF loading for the 31 antibodies is 4.42 andthe coefficient of variation falls to 3.45%. These results illustratethe consistency of reagent loading (mcMMAF and AF647) achieved by thepresent method across a panel of antibodies of variable isotype and invariable quantities from a panel of newly fused hybridomas.

Example 6 Exemplary Method for Making Antibody Conjugates Loaded withDrug-Linker and Capping Agent

Hybridoma supernatants were prepared as 4.5 mL solutions in 5 mLround-bottom tubes. 150 uL Protein G resin slurry (Millipore ProSep-G)was added to each. Tubes were capped and rotated overnight at 5° C. Twocontrol tubes were also prepared, one with bovine IgG-depleted growthmedia (to serve as a blank) and one with this same media spiked with 100ug of a control antibody.

On the following morning, resin was transferred from tubes to a 2 mL,96-well filter plate with a 2.5 um polypropylene frit (SeahorseBioscience) using a 1250 uL Matrix pipettor. The supernatant in thefilter plate was pulled through by brief application of vacuum. Afterall wells had dried (<30 seconds), the plate was centrifuged at 500×gfor 3 minutes to ensure complete pulldown of all fluids and resin. Afterspinning, the filter plate was replaced on the manifold and each wellreceived 500 uL PBS. The plate was then shaken at 1200 RPM on theThermomixer for 30 seconds to slurry the resin. The PBS was then pulledthrough by vacuum. This process was repeated twice, for a total of 3 PBSwashes. This process was then repeated with 3x PBS, and then followed bya another wash with PBS. Following this final wash, the plate was spunas before.

The bound antibodies were then reduced by adding 500 uL of 20 mM TCEP in250 mM KPO₄, 150 mM NaCl, pH 7, 1 mM EDTA and shaking for 2 hours at 37°C. on the Thermomixer. Following reduction, the TCEP solution was pulledthrough by vacuum and then spun as above, then washed with PBS+1 mM EDTAas described above. This was repeated 4×, for a total of 5 washes.

The bound antibodies were then conjugated to a mixture of NEM andmc-MMAF in a molar ratio of 4:6. A stock of NEM+mc-MMAF at a totalmaleimide concentration of 12 mM was prepared in advance. 1.1 mL of thissolution was added to 55 mL of 10% DMSO, transferred to a multichannelreservoir, and 500 uL added to each well of the filter plate, which wasthen shaken for 30 minutes at 22° C. Following conjugation, themaleimide solution was pulled through by vacuum and then spun as above.The centrifuge speed was increased to 1500×g to complete the drying.Wells were then washed twice with 500 uL of 10% DMSO in PBS, then threetimes with PBS.

The bound ADCs were then eluted by adding 200 uL of 100 mM glycine, pH2.0 to each well and shaking for 3 minutes at 1200 RPM, 22° C. on theThermomixer. While shaking, 20 uL of neutralization buffer (1M dibasicphosphate+0.1% Tween-20) was added to each well of a 350 uL collectionplate. When 3 minutes had elapsed, the ADCs were eluted into thecollection plate by spinning at 1500×g for 6 minutes.

200 uL of each ADC solution were transferred to a Costar UV assay plate.A second plate was prepared with neutralized elution buffer to serve asa blank. A280 measurements were carried out with a Molecular DevicesSpectraMax plate reader to determine protein concentrations.

Finally, ADCs were sterile filtered. In the BSC, a sterile 0.2 um filterplate (Millipore) was fastened to a sterile 1 mL collection plate(Matrix) using lab tape. The ADC solutions were then added to the filterplate and spun at 500×g for 3 minutes. The assembly was then transferredto the BSC and disassembled, then the collection plate capped with asterile cap mat (Matrix).

Example 7 Exemplary Method for Making Antibody Conjugates Loaded withDrug-Linker, Capping Agent, and Fluorophore

Newly fused hybridomas were plated in methylcellulose media (Genetix)containing HAT and fluorescently labeled anti-mouse IgG (Genetix).Clonal, IgG-producing colonies were selected and deposited into a 96 Wplate containing HSFM (InVitrogen) plus IgG-depleted cloning factor(Roche). Four-fold dilutions of unpurified hybridoma culturesupernatants were incubated with target tumor cells in a homogenousassay containing 100 ng/ml of Cy5-labeled anti-mouse secondary antibody(Jackson Labs). Hybridoma binding to the tumor cells was detected usingan FMAT8200 (Applied Biosytems) and positive wells were expanded into 48W dishes containing 2 mls of HSFM (InVitrogen) plus IgG-depleted cloningfactor. Antibodies from 48 W extinguished supernatants were used forsolid-phase purification and conjugation.

Hybridoma supernatants (1.5 mL) were transferred to 96-well deep wellplates with a 0.45 um polypropylene frit (Seahorse Bioscience). Toenable quantitation of conjugate concentration by fluorescence, astandard murine antibody was included in the conjugation. 50 ug of thestandard antibody was placed in 4 wells of the plate with blank media(200 ug total). Additionally, 3 wells contained only blank media fordetermination of background fluorescence.

100 uL of PBS was placed in each well of a 96-well deep well filterplate fitted with a 2.5 um polypropylene filter (Seahorse Bioscience).20 uL Protein G resin slurry (GE Life Sciences GammaBind Plus) was addedto each well.

The filter plate containing the Protein G resin was placed as thereceiver plate in a vacuum manifold, and the manifold assembled. The0.45 um filter plate containing the antibody samples and standards wasplaced on top of the manifold, and the supernatants transferred to it.By application of vacuum, supernatants were then filtered through the0.45 um filters into the plate containing the Protein G resin. The resinplate was then shaken for 2 hours at room temperature at 1200 RPM usingan Eppendorf Thermomixer to effect binding to the Protein G. Theresidual supernatant was then filtered into a 2 mL deep well receiverplate by centrifugation at 500×g for 5 minutes.

A solution of 10 mM tricarboxyethyl phosphine (TCEP) in 100 mM potassiumphosphate, pH 7.4, 150 mM NaCl, was added to the plate (150 uL perwell). The plate was then shaken as above for 30 minutes, then removedfrom the shaker and centrifuged for 2 minutes at 500×g. The resin waswashed four times with 500 uL of PBS containing 1 mM EDTA, with vacuumfiltration following each wash. Following the final wash, another 500 uLPBS/EDTA added and removed by centrifugation for 3 minutes at 500×g.

Individual stocks of drug-linker (mcMMAF), Alexa Fluor® 647, and NEMwere prepared at 10 mM in DMSO. These stocks were then blended into asingle solution at the following ratio for conjugation:

3:3:1 mcMMAF:Alexa Fluor® 647:NEM

140 uL of this solution was dissolved in 15 mL of PBS/EDTA, and 150 uLadded to each well of the washed plate. The plate was then shaken asabove for 15 minutes, then removed from the shaker and centrifuged for 2minutes at 500×g. The resin was washed four times with 500 uL of PBS,with vacuum filtration following each wash. Following the final wash,another 500 uL of PBS was added and removed by centrifugation for 3minutes at 500×g.

10 uL of 1M potassium phosphate pH 7.4 was added to each well of a 350uL 96-well clear-bottom assay plate. The resin plate was placed atop theassay plate and 100 uL elution buffer (50 mM glycine pH 2.5+0.08%Tween-20) added to each well. The plate was gently agitated by manualrocking for 2 minutes, then centrifuged for 2 minutes at 500×g tocollect the eluted antibody conjugates in the assay plate. The assayplate was immediately placed in a fluorescence plate reader (MolecularDevices) and shaken for 10 seconds using the plate reader shaker toensure complete mixing of the neutralization buffer into the elutionbuffer. The fluorescence of each well at 675 nm was then measured usingan excitation of 635 nm with a 665 nm cutoff filter. The solutions inthe wells containing the standards were removed and pooled into a singlestandard solution, and the concentration of this standard was determinedby a conventional A280 absorbance method in a 1 cm cuvet. A dilutionseries of this standard was then prepared (using neutralized elutionbuffer as the diluent) down to a concentration of 1 ug/mL. 110 uL ofeach standard was then transferred to a clean 350 uL clear bottom assayplate and the fluorescence again measured on the plate reader. Asecond-order polynomial curve was fit to the fluorescence values of thestandards, and the concentrations of the samples were assigned byinterpolation to this standard curve.

Finally, ADCs were sterile filtered. In the BSC, a sterile 0.2 um filterplate (Millipore) was fastened to a sterile 1 mL collection plate(Matrix) using lab tape. The ADC solutions were then added to the filterplate and spun at 500×g for 3 minutes. The assembly was then transferredto the BSC and disassembled, then the collection plate capped with asterile cap mat (Matrix).

Mixed antibody conjugates containing fluorophore and drug were tested incell binding and cytoxicity assays. For cell-based binding assays, theantibody panel was diluted at 1:200 and 1:1000 in PBS+2% serum andincubated on target cells for 2 hours at room temperature in 96 W blackplates. A control antibody was used on each plate to generate asaturation binding curve for human and cyno forms of the antigen. Plateswere then analyzed in an FMAT8200 and mean fluorescence intensity valuesfor each dilution were plotted on the saturation binding curve toestimate test antibody affinity on human and cyno forms of the antigen.Hybridomas that showed equivalent binding to human and cyno antigen wereadvanced for cytotoxicity studies. Cytoxicity studies were done byplating 5,000 cells per well in the appropriate growth media. Mixedconjugates were added to a final dilution of 1:100 and 1:1000,respectively. Tumor cells were incubated with drug/fluorophoreconjugates for 96 hours at 37° C. Cell Titer Glo (Promega) was used tomeasure cell viability and the potency of drug/fluorophore conjugateswas assessed based on the percent viability relative to untreatedcontrol cells. Drug/fluorophore conjugates that resulted in <70%viability of tumor cells at 1 nM concentrations were advanced forfurther testing.

Example 8 IgG Depletion

Cloning Factor is commonly used as a media component in expandinghybridoma cell lines after fusion with murine B cells. Cloning Factorcontains important cell mediators that where harvested from thesupernatant of healthy thriving cells and these help the new hybridomafusions recover and begin to grow more robustly. It is suspected thatthe harvested supernatant from the healthy thriving cells that makes upthe cloning factor contains bovine serum as a media component whichwould include bovine albumin, IgG and other serum proteins. It is thebovine IgG that is of concern in this case because even a small amountof contaminating IgG can affect the quantitative recovery of theantibodies and quantitation of the resulting ADCs.

The method for removing bovine IgG from Cloning Factor is as follows. 5ml Protein G column is equilibrated with 1× PBS (5 Column Volumes, CV),25 ml. Contents of the Hybridoma Cloning factor are loaded into a 60 ccsyringe. A syringe is attached to the Protein G column and connected toa syringe pump. The pump is set to 3 ml/min, the Cloning Factor ispassed over the Protein G column and the effluent is collected. Theeffluent contains the IgG depleted Cloning factor. Bovine IgG will bindto the Protein G column. The IgG depleted Hybridoma Cloning factor issterile filtered in a biosafety cabinet using a 0.22 μm syringe filter.

What is claimed:
 1. A method for making antibody conjugates for use inantibody screening assays, the method comprising the steps of: (a)providing a first and second antibody-containing sample, wherein thefirst and second antibody-containing samples vary with respect toantibody quantity and antibody sequence provided that substantially allof the antibody present in the first sample is of the same sequence andsubstantially all of the antibody present in the second sample is of thesame sequence, wherein the antibodies of the first and second sampleshave sulfhydryl groups introduced into said antibodies; (b) immobilizingthe antibodies on solid support to provide a first and second samplecomprising immobilized antibodies; (c) fully reducing the interchaindisulfide bonds and introduced sulfhydryl groups of the immobilizedantibodies to provide a first sample comprising reduced immobilizedantibodies and second sample comprising reduced immobilized antibodies;(d) reacting the reduced immobilized antibodies with capping agent, drugor drug-linker, and optionally a detection agent to provide immobilizedantibody conjugates, wherein the capping agent, drug or drug-linker, andoptional detection agent selectively react with reactive thiols, thecapping agent, drug or drug-linker, and optional detection agent areprovided in molar excess, and the ratio of capping agent, drug or druglinker, and optional detection agent is selected so as to achieve adesired level of drug loading; and (e) eluting the antibody conjugatesto provide a first and second antibody drug conjugate composition. 2.The method of claim 1 wherein the antibody in the first and secondantibody-containing samples have the same number of introducedsulfhydryl groups and the reduced immobilized antibodies are contactedwith the same ratio of capping agent, drug or drug-linker, and optionaldetection agent.
 3. The method of claim 1 wherein the antibody in thefirst and second antibody-containing samples have different number ofintroduced sulfhydryl groups and the reduced immobilized antibodies arecontacted with a different ratio of capping agent, drug or drug-linker,and optional detection agent.
 4. The method of claim 1 wherein thereduced immobilized antibodies are reacted with a detection agent. 5.The method of claim 4 wherein the detection agent is a fluorophore andthe resultant antibody conjugates compositions have an averagefluorophore loading of about 3 fluorophores per antibody.
 6. The methodof claim 1 wherein following elution of the antibody conjugates, theactual or relative quantity of antibody present in the antibodyconjugate compositions is determined.
 7. The method of claim 1 whereinthe antibody present in the antibody-containing samples prior toimmobilization is impure.
 8. The method of claim 1 wherein the quantityof antibody present in the antibody-containing samples prior toimmobilizing, reduction, conjugation, and elution is not known.
 9. Themethod of claim 1 wherein the antibody-containing samples independentlyhave from 1 μg to 100 μg of antibody present in each of the sample. 10.The method of claim 9 wherein the antibody-containing samplesindependently have from 1 μg to 50 μg of antibody present in each of thesamples.
 11. The method of claim 10 wherein the antibody containingsamples independently have from 1 μg to 20 μg of antibody present ineach of the samples.
 12. The method of claim 1 wherein the antibody inthe first antibody-containing sample and the antibody in the secondantibody-containing sample are of the same species.
 13. The method ofclaim 1 wherein the capping agent, drug or drug-linker and detectionagent comprise a maleimide group.
 14. The method of claim 13 wherein thedrug-linker loading between samples is substantially uniform.
 15. Themethod of claim 14 wherein the antibody conjugate compositions have anaverage drug-linker loading of about 2 drug-linkers per antibody. 16.The method of claim 1 wherein the antibody-containing samples are cellculture supernatant samples.
 17. The method of claim 16 wherein the cellculture supernatant is unpurified hybridoma cell culture supernatant.18. The method of claim 16 wherein the cell culture supernatant isunpurified CHO cell culture supernatant.
 19. The method of claim 16wherein the cell culture media used for providing theantibody-containing samples was substantially IgG depleted.
 20. Themethod of claim 16 wherein the solid support is added to the cellculture supernatant.
 21. A method for making antibody conjugates for usein high throughput screening assays comprising the steps of: (a)providing a plurality of samples of unpurified hybridoma supernatantcomprising unquantified antibody produced from a plurality of hybridomaclones, wherein the plurality of samples vary with respect to antibodyquantity and antibody sequence provided that, in a majority of theplurality of the samples, substantially all of the antibody present ineach sample is from a single hybridoma clone wherein the antibodies ofthe plurality of samples have sulfhydryl groups introduced into saidantibodies; (b) immobilizing the unquantified antibodies on solidsupport to provide a plurality of samples comprising immobilizedantibodies; (c) fully reducing the intrachain disulfide and introducedsulfhydryl groups of the immobilized antibodies to provide a pluralityof samples comprising reduced immobilized antibodies; (d) reacting thereduced immobilized antibodies with capping agent, drug or drug-linker,and a detection agent to provide immobilized antibody conjugates,wherein the capping agent, drug or drug-linker, and detection agentselectively react with reactive thiols, the capping agent, drug ordrug-linker, and optional detection agent are provided in molar excess,and the ratio of capping agent, drug or drug linker and detection agentis selected so as to achieve a desired level of drug loading; and (e)eluting the immobilized antibody conjugates from the solid supports toprovide a plurality of antibody conjugate compositions.
 22. The methodof claim 21 wherein the cell culture media used for production of theantibody-containing samples was substantially IgG depleted.
 23. Themethod claim 21 wherein there is from 1 μg to 100 μg of antibody presentin each sample of hybridoma supernatant.
 24. The method claim 23 whereinthere is from 1 μg to 50 μg of antibody present in each sample ofhybridoma supernatant.
 25. The method of claim 24 wherein there is from1 μg to 20 μg of antibody present in each sample of hybridomasupernatant.
 26. The method of claim 21 wherein the detection agent is afluorescent label.
 27. The method of claim 21 wherein the capping agent,drug or drug-linker and detection agent comprise a maleimide group. 28.The method of claim 21 wherein the drug-linker loading between samplesis substantially uniform.
 29. A method for preparing antibodyconjugates, the method comprising the steps of: (a) providing aplurality of antibody-containing samples that vary with respect toantibody quantity and antibody sequence provided that, in a majority ofthe plurality of the antibody-containing samples, substantially all ofthe antibody present in a single sample is of the same sequence; (b)immobilizing the antibodies on solid support to provide a plurality ofsamples comprising immobilized antibodies; (c) fully reducing theinterchain disulfide bonds and introduced sulfhydryl groups of theimmobilized antibodies to provide a plurality of samples comprisingreduced immobilized antibodies, (d) reacting the reduced immobilizedantibodies with capping agent, drug or drug-linker, and optionally adetection agent to provide a plurality of samples comprising immobilizedantibody conjugates, wherein the capping agent, drug or drug-linker, andoptional detection agent selectively react with reactive thiols, thecapping agent, drug or drug-linker, and optional detection agent areprovided in molar excess, and the ratio of capping agent, drug or druglinker; and optional detection agent is selected so as to achieve adesired level of drug loading; and (e) eluting the antibody conjugatesto provide a plurality of antibody conjugate compositions.
 30. A methodfor selecting an antibody for use in an antibody drug conjugate, themethod comprising the steps of: (a) providing a plurality of antibodycontaining samples that vary with respect to antibody quantity andantibody sequence provided that, in a majority of the plurality of theantibody-containing samples, substantially all of the antibody presentin a single sample is of the same sequence; (b) immobilizing theantibodies on a solid support to provide a plurality of samplescomprising immobilized antibodies; (c) fully reducing the intrachaindisulfide bonds and introduced sulfhydryl groups of the immobilizedantibodies to provide a plurality of samples comprising reducedimmobilized antibodies; (d) reacting the reduced immobilized antibodieswith capping agent, drug or drug-linker, and optionally a detectionagent to provide a plurality of samples comprising immobilized antibodyconjugates, wherein the capping agent, drug or drug-linker, and optionaldetection agent selectively react with reactive thiols, the cappingagent, drug or drug-linker, and optional detection agent are provided inmolar excess, and the ratio of capping agent, drug or drug linker; andoptional detection agent is selected so as to achieve a desired level ofdrug loading; (e) eluting the antibody conjugates to provide a pluralityof antibody conjugate compositions comprising free antibody conjugates;(f) assaying for an activity of the antibody conjugates; (g) determiningthe actual or relative quantity of antibody present in the antibodyconjugate compositions; (h) selecting the antibody providing antibodydrug conjugates with the greatest activity based on the results of theassay and the actual or relative quantity of antibody present in theantibody conjugate compositions.
 31. The method of claim 30, wherein theactivity is cytotoxicity.